Methods and compositions for activation of innate immune responses through RIG-I like receptor signaling

ABSTRACT

Compositions and methods are provided that enable activation of innate immune responses through RIG-I like receptor signaling. The compositions and methods incorporate synthetic nucleic acid pathogen associated molecular patterns (PAMPs) that comprise elements initially characterized in, and derived from, the hepatitis C virus genome.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.61/844,022, filed Jul. 9, 2013, which is incorporated herein byreference in its entirety.

STATEMENT OF GOVERNMENT LICENSE RIGHTS

This invention was made with Government support under AI100384,DA024563, AI088788, and AI060389 awarded by the National Institutes ofHealth. The Government has certain rights in the invention.

STATEMENT REGARDING SEQUENCE LISTING

The sequence listing associated with this application is provided intext format in lieu of a paper copy and is hereby incorporated byreference into the specification. The name of the text file containingthe sequence listing is 50077_SEQ_ST25.txt. The text file is 26 KB; wascreated on Jul. 8, 2014; and is being submitted via EFS-Web with thefiling of the specification.

BACKGROUND

The innate immune system is often the first line of defense againstcolonization and/or proliferation by invading pathogens, such as virusesand bacteria. The innate immune system comprises cells and circulatingcomponents that, upon detection of a non-self entity (such as apathogen) within the body, can act non-specifically to counter theinvasion. Various effects of the innate response can include therecruitment of phagocytic cells to sites of infections throughproduction of chemokines and the promotion of inflammation.Additionally, the innate response can include the activation of serumcomplement factors to damage pathogen membranes. Moreover, the innateresponse can include the production of cytokines, such as interferons,to induce antiviral states of uninfected cells. For example, the rapidproduction of alpha/beta interferon (IFN α/β) leads to the inducedexpression of hundreds of interferon-stimulated genes (SGs) whoseproducts direct anti-pathogen and immunomodulatory actions that cancounter-act infections. While the mechanisms of the innate system aregenerally non-specific and short-lived, the innate immune system alsocross-activates elements of the adaptive immune system, which canrespond to specific foreign antigens through the antigen-specificinteractions of antibodies and TCR receptors. For example, macrophagesthat encounter foreign pathogens can produce various cytokines thatcontribute to the activation of various components of the adaptiveimmune system.

Appropriate innate immune responses only occur upon detection ofnon-host pathogens and limit the severity of the response to avoid unduedamage to healthy host tissue (e.g., avoid septic shock). Non-hostpathogens can be detected by the discrimination between host (self) andnon-self antigens. Various classes of pathogens, such as viruses andbacteria, contain pathogen-associated molecular patterns (PAMPs) instructural or genetic components that are not exhibited by hostorganisms. Most mammalian cells have receptors that recognize PAMPScalled pattern recognition receptors (PRRs), which, when bound to theappropriate PAMPs will signal the presence of non-host organisms.However, considering the general, non-specific response of the innateimmune systems, accurate and appropriate activation of the innateresponse to pathogen patterns instead of host patterns is essential toavoid causing damage to host tissues. Furthermore, the appropriatedegree of innate response is also critical because innate responses arenot antigen specific and damage to host tissue can result fromoverstimulation. Such damage can be more costly to the host organismthan the infection itself. In extreme cases, hosts can experience septicshock when the innate immune system is overstimulated.

Identifying PAMPs that induce innate immune response can be useful toserve as anti-microbial therapeutics, such as adjuvants in combating orpreventing infections, and to enhance the efficacy of more traditionalvaccine therapeutics. Accordingly, there is a need to identifypathogen-associated molecular patterns (PAMPs) that can stimulate anappropriate and effective innate immune response to a pathogen but thatcan avoid costly damage to tissues of the host organism. Thecompositions and methods of the present disclosure address this andrelated needs.

SUMMARY

This summary is provided to introduce a selection of concepts in asimplified form that are further described below in the DetailedDescription. This summary is not intended to identify key features ofthe claimed subject matter, nor is it intended to be used as an aid indetermining the scope of the claimed subject matter.

In one aspect, the disclosure provides a synthetic nucleic acidpathogen-associated molecular pattern (PAMP). In one embodiment, thesynthetic nucleic acid PAMP comprises a 5′-arm region, a poly-uracilcore, and a 3′-arm region.

In one embodiment, the 5′-arm region comprises a terminal triphosphate.In one embodiment, the 5′-arm region further comprises one or morenucleic acid residues disposed between the terminal triphosphate and thepoly-uracil core.

In one embodiment, the poly-uracil core comprises at least 8 contiguousuracil residues. In one embodiment, the poly-uracil core consists ofbetween 8 and 30 uracil residues.

In one embodiment, the 3′ arm region comprises at least 8 nucleic acidresidues. In one embodiment, the 5′ most nucleic acid residue of the 3′arm region is not a uracil and the 3′ arm region is at least 30% uracilresidues. In one embodiment, the 5′-most nucleic acid residue of the3′-arm region is a cytosine residue or a guanine residue. In oneembodiment, the 3′-arm region is at least 40%, 50%, 60%, 70%, 80%, or90% uracil residues. In one embodiment, the 3′-arm region comprises atleast 7 contiguous uracil residues.

In one embodiment, the terminal triphosphate, the one or more nucleicacid residues of the 5′-arm region, and the poly-uracil core do notnaturally occur together in a Hepatitis C virus.

In one embodiment, the synthetic nucleic acid PAMP is capable ofinducing retinoic acid-inducible gene I (RIG-I)-like receptor (RLR)activation. In one embodiment, the RLR is RIG-I.

In another aspect, the disclosure provides a pharmaceutical composition.In one embodiment, the pharmaceutical composition comprises thesynthetic nucleic acid PAMP described herein and an acceptable carrier.In one embodiment, the pharmaceutical composition further comprises aviral antigen, a bacterial antigen, a protozoal antigen, a fungalantigen, and/or a helminth antigen, or an attenuated, inactivated, orkilled virus, bacterium, protozoan, fungus, and/or helminth. In oneembodiment, the pharmaceutical composition further comprises ananti-viral therapeutic, an anti-bacterial therapeutic, an anti-protozoaltherapeutic, an anti-fungal therapeutic, an anti-helminth therapeutic,and/or an adjuvant.

In another aspect, the disclosure provides a method of inducing retinoicacid-inducible gene I (RIG-I)-like receptor (RLR) signaling in a cell.In one embodiment, the method comprises administering to the cell aneffective amount of the synthetic nucleic acid PAMP described herein.

In another aspect, the disclosure provides a method of treating acondition in a subject treatable by inducing RLR signaling. In oneembodiment, the method comprises administering to the subject aneffective amount of the pharmaceutical composition described herein.

In another aspect, the disclosure provides a method of inducing aninnate immune response in a subject. In one embodiment, the methodcomprises administering to the subject an effective amount of thepharmaceutical composition described herein.

In another aspect, the disclosure provides a method of treating a viral,bacterial, protozoal, fungal, and/or helminth infection in a subject. Inone embodiment, the method comprises administering to the subject aneffective amount of the pharmaceutical composition described herein.

DESCRIPTION OF THE DRAWINGS

The foregoing aspects and many of the attendant advantages of thisinvention will become more readily appreciated as the same become betterunderstood by reference to the following detailed description, whentaken in conjunction with the accompanying drawings, wherein:

FIGS. 1A-1C illustrate that HCV-derived poly-U/UC RNA constructsactivate RIG-I signaling. (FIG. 1A) Induction of the IFN-β-promoter inHuh7 cells transfected with equal moles of tRNA, full-length JFH1, JFH1pU/UC, or Con1 pU/UC RNA. IFN-β-promoter luciferase activity is shown asmean IFN-β fold index (compared to cells with No RNA, ±s.d. for threereplicates). Huh7 cells were transfected with the various RNA constructsand 16 hours later cells were harvested for dual luciferase activity.Asterisks indicate a significant difference compared to No RNA controlas determined by a one-way ANOVA adjusted with Bonferroni's multiplecomparison test (*P<0.05, **P<0.01, ***P<0.001). (FIG. 1B) Induction ofthe IFN-β-promoter in Huh7 or Huh7.5 cells transfected with 350 ng ofthe indicated RNA constructs (described in more detail in Example 1).IFN-β-promoter luciferase activity is shown as the mean IFN-β fold index±s.d. for three replicates, and data was normalized to the No RNAcontrol. Cells were harvested for dual luciferase activity 16 hourspost-RNA transfection. Asterisks indicate a significant differencecompared to No RNA control as determined by a one-way ANOVA adjustedwith Bonferroni's multiple comparison test (*P<0.05, **P<0.001). (FIG.1C) The abundance of phospho-IRF-3 (Ser396), total IRF-3, RIG-I, ISG56,and tubulin were measured by immunoblot. Huh7 cells were transfectedwith the indicated RNA constructs and cells were harvested for proteinanalysis 16 hours later. RIG-I and ISG56 are IFN-β-stimulated genes. Theratio of phospho-IRF-3/total IRF-3 was calculated by measuring therelative immunoblot band intensities using ImageJ software (NIH). Datashown in all panels are representative of three independent experiments.

FIGS. 2A-2C illustrate the differential binding between poly-U/UC RNAconstructs and purified RIG-I protein in vitro. (FIG. 2A) EMSA gel-shiftassays: RNA (10 pmol) was incubated with increasing concentrations ofpurified recombinant RIG-I protein (0-30 pmol), then complexes wereseparated on native agarose gels and RNA was visualized using SYBR Goldnucleic acid stain. Unshifted RNA, U; shifted RNA/protein complex, S;supershifted RNA/protein complex, ss. (FIG. 2B) RIG-I/RNA binding curveswere generated from the gel-shift analyses and plotted for the X-region,X-region-U34, Con1 pU/UC, JFH1 pU/UC, Δcore, U8core, U17core, and poly-U62-C RNAs. Due to poor band formation of the poly-U 62 RNA on thenon-denaturing gel used in our EMSA, a gel-shift analysis of thisparticular RNA could not be conducted. (FIG. 2C) Table comparing theeffective pmol concentration of RIG-I required to shift 10% (EC₁₀), 50%(EC₅₀), or 90% (EC₉₀) of each RNA construct. RIG-I signaling for eachRNA construct was determined in FIG. 1B, and the magnitude of thesignaling activity is listed in the table as either None/Low (IFN-β foldindex=0-3), Low (IFN-β fold index=3-30), Med. (IFN-β fold index=30-100),or High (IFN-b fold index >100). N/A=Not applicable.

FIGS. 3A-3C illustrate that HCV poly-U/UC RNA constructs interact withthe RIG-I RD and helicase domain. (FIG. 3A) Limited-trypsin proteolysisof 30 pmol purified RIG-I with increasing amounts of RNA. Repressordomain, RD; helicase domain and CARDs, Helic. +CARDs. (FIG. 3B) Limitedtrypsin proteolysis of 30 pmol purified RIG-I protein with 1.0 pmol ofeach indicated RNA construct. RIG-I digestion products were separated onthe same gel and relative band intensities (listed as % of total) weremeasured using ImageJ gel imaging software (NIH). (FIG. 3C) ATPaseactivity of purified RIG-I protein incubated with increasing amounts ofRNA. Data shown are means±s.d. for two replicates.

FIGS. 4A-4C illustrate that HCV poly-U/UC RNA variants triggerdifferential anti-HCV and hepatic innate immune responses. (FIG. 4A)Huh7 cells were transfected with the indicated poly-U/UC RNA constructs12 hours prior to HCV infection (MOI=0.1), and virus production wasassessed 48 hours post-infection. Data shown are means±s.d. for threereplicates. Asterisks indicate a significant difference compared to NoRNA control as determined by a one-way ANOVA adjusted with Bonferroni'smultiple comparison test (*P<0.001, **P≦0.0001). (FIG. 4B) Wild-typemice (n=2) received 200 μg of X-region RNA, X-region-U34 RNA, Con1 pU/UCRNA, or Δcore RNA. Mock-transfected wild-type mice (n=1) received PBS.Comparative measurements of hepatic mRNA and protein expression weremeasured 8 hours post-transfection. Real-time quantitative PCR wasperformed to examine expression of IFN-β, CCL5, Ifit2, ISG15, and GAPDH.Results were normalized to the expression of mouse GAPDH mRNA, and mRNAfold index was normalized to Mock controls. See the left panel for IFN-βand CCL5 expression and the right panel for Ifit2 and ISG15 expression.Data shown are means±s.d. for two replicates, and gene expression datawas confirmed by two independent real-time PCR analyses. Asterisksindicate a significant difference as determined by a one-way ANOVAadjusted with Bonferroni's multiple comparison test (*P<0.05, **P<0.01,***P<0.001). (FIG. 4C) Following RNA transfection, mouse livers wererecovered and immunohistochemistry staining was conducted for mouseISG54. The black scale bar indicates a distance of 500 mm.

FIGS. 5A and 5B illustrates that IFN-beta promoter is induced bypoly-U/UC, U12, U17, and U24 core RNA constructs. Interferon-betaluciferase activity was measured in Huh7 cells transfected with 10⁵cells were co-transfected with 200 ng of the indicated RNA constructderived from HCV (Con1 strain), along with IFN-beta luc reporterpromoter plasmid. Cells were harvested 16 hrs later. FIG. 5A illustratesthe relative IFN-β luciferase activity stimulated by the various RNAconstructs. FIG. 5B illustrates an immunoblot of cells harvested inparallel and assayed for abundance of total IRF-3, Phospho-IRF-3(P-IRF-3), and actin.

FIG. 6 illustrates the antiviral activity of the poly-U/UC PAMP RNAconstruct. Abbreviations: JEV, Japanese encephalitis virus; VSV,vesicular stomatitis virus; DV, dengue virus 2; WNV, West Nile virus;HCV, hepatitis C virus; RSV, respiratory syncytial virus. The graphshows viral load in cultured Huh7 (human hepatoma) cultures treated withtransfection reagent alone (white) and in cultures transfected withpoly-U/UC RNA (black). Cells were infected with virus at MOI=1.0, andafter 3 hrs were treated as indicated. Cells were harvested at 72 hourspost-treatment and intracellular viral RNA levels measured byvirus-specific reverse transcriptase-quantitative PCR (RT-qPCR) assay.Bars show intracellular viral RNA levels compared to transfectionreagent control. Error bars show standard error of the mean across 6experiments. Differences between each control and poly-U/UC bar set aresignificant (P<0.03) based on Student's T-test. This demonstrates thatthe poly-U/UC RNA construct has antiviral activity.

FIG. 7 illustrates the antiviral activity of U 17 core PAMP RNAconstruct. Abbreviations: JEV, Japanese encephalitis virus; VSV,vesicular stomatitis virus; DV, dengue virus 2; WNV, West Nile virus;HCV, hepatitis C virus; RSV, respiratory syncytial virus. The graphshows viral load in cultured Huh7 (human hepatoma) cultures treated withtransfection reagent alone (white) and in cultures transfected with theU17 core derivative of the poly-U/UC RNA PAMP (black). Cells wereinfected with virus at MOI=1.0, and after 3 hrs were treated asindicated. Cells were harvested at 72 hours post-treatment andintracellular viral RNA was measured by virus-specific RT-qPCR assay.Bars show intracellular viral RNA level compared to control. Error barsshow standard error of the mean across 4 experiments. Differencesbetween each control and U17 core RNA bar set are significant (P<0.01)based on Student's T-test. This demonstrates that the U 17 core RNA hasantiviral activity.

FIG. 8 illustrates the antiviral activity of U12 core PAMP RNAconstruct. Abbreviations: JEV, Japanese encephalitis virus; VSV,vesicular stomatitis virus; DV, dengue virus 2; WNV, West Nile virus;HCV, hepatitis C virus; RSV, respiratory syncytial virus. The graphshows viral load in cultured Huh7 (human hepatoma) cultures treated withtransfection reagent alone (white) and in cultures transfected with theU 12 core derivative of the poly-U/UC RNA PAMP (black). Cells wereinfected with virus at MOI=1.0, and after 3 hrs were treated asindicated. Cells were harvested at 72 hours post-treatment andintracellular viral RNA was measured by virus-specific RT-qPCR assay.Bars show intracellular viral RNA level compared to control. Error barsshow standard error of the mean across 3 experiments. Differencesbetween each control and U12 core RNA bar set are significant (P<0.05)based on Student's T-test. This demonstrates that the U12 core RNA hasantiviral activity.

FIGS. 9A-9C illustrate that injection of polyU/UC RNA induces the innateimmune response in vivo through MAVS/RLR-mediated signaling. 200 mgpolyU/UC RNA or PBS control was administered to wildtype and MAVS −/−mice (MAVS−/− mice lack the MAVS adaptor protein that mediatesRIG-1-like receptor (RLR) signaling) by hydrodynamic IV injection in thetail vein or IP. Mice were euthanized 8 hrs later and assessed for (FIG.9A) ISG54 expression in the liver by immunohistochemistry, (FIG. 9B)innate immune antiviral gene induction in the liver by immunoblot and(FIG. 9C) IFNb levels in the sera by ELISA.

FIG. 10 illustrates that HCV polyU/UC RNA treatment reduces HCV viralburden in vivo. SCID/beige-Alb/uPA chimeric mice were transplanted withhuman primary hepatocytes derived from cryopreserved stocks purchasedfrom CellDirect Inc. Human hepatocyte repopulation levels were verifiedat 4 and 8 weeks after transplantation by measuring human albumin levelsby ELISA. Chimeric animals with human albumin concentrations greaterthan or equal to 1000 mg/mL received a single intravenous injection of100 mL HCV-positive patient serum (HCV genotype 2b, 1.38×10⁵ genomeequivalents per animal). Mice were administered PBS alone or 150 mgpolyU/UC (PAMP) RNA or control xRNA by hydrodynamic IV injections viathe tail vein at days 11, 13 and 15 post-infection. Infected mice werebled at days 7 (pre-treatment baseline) and 14 post-infection forviremia measurements of HCV genomic RNA by qPCR. Shown here isdifference in viral burden (log scale) in the sera compared to the day 7baseline.

FIGS. 11A-11B illustrate that HCV polyU/UC RNA treatment reduces HCVviral burden in vivo. SCID/beige-Alb/uPA chimeric mice were transplantedwith human primary hepatocytes derived from cryopreserved stockspurchased from CellDirect Inc. Human hepatocyte repopulation levels wereverified at 4 and 8 weeks after transplantation by measuring humanalbumin levels by ELISA. Chimeric animals with human albuminconcentrations greater than or equal to 1000 mg/mL received a singleintravenous injection of 100 mL HCV-positive patient serum (HCV genotype2b, 1.38×10⁵ genome equivalents per animal). Mice were administered PBSalone or 150 mg polyU/UC (PAMP) RNA or control xRNA by hydrodynamic IVinjections via the tail vein at days 11, 13, and 15 post-infection.Infected mice were bled at days 10 (pre-treatment baseline) and at days14 and 15 post-infection for viremia measurements of HCV genomic RNA byqPCR. Illustrated here are differences in viral burden (log scale) inthe sera on day 14 (FIG. 11A) and day 15 (FIG. 1B) compared to the day10 baseline.

FIG. 12 illustrates that polyU/UC RNA, but not xRNA treatment, protectsmice against disease from West Nile virus (WNV) infection. C57Bl6 micewere infected via subcutaneous injection of the left footpad with 1000pfu WNV (Tx-02). At days 1.5, 3, and 5, mice were given 100 mg polyU/UCRNA or control xRNA by IP. Mice were monitored daily for body weight andclinical scores. Mice that lose greater than or equal to 20%/a bodyweight or that exhibit clinical symptoms greater than 5 were euthanizedaccording to UW IACUC protocols. The graph shows percentsurvival/morbidity curve for mice over a course of 17 dayspost-infection. This demonstrates that poly-U/UC RNA providestherapeutic protection against WNV infection.

FIGS. 13A-13D illustrate that polyU/UC RNA treatment protects mice afterWest Nile virus (WNV) infection in a MAVS/RLR signaling-dependentmanner. Wildtype and MAVS −/− mice were infected via subcutaneousinjection of the left footpad with 1000 pfu WNV (Tx-02). At days 1.5, 3,and 5, mice were given 200 mg polyU/UC RNA or PBS control byhydrodynamic IV injection in the tail vein. Mice were monitored dailyfor body weight and clinical scores. Mice that lose greater than orequal to 20% body weight or that exhibit clinical symptoms greater than5 were euthanized according to UW IACUC protocols. FIG. 13A shows thepercent survival/morbidity curve, FIG. 13B shows the average bodyweight, and FIG. 13C shows the average clinical score for mice over acourse of 17 days post-infection. FIG. 13D shows results of a standardplaque assay in a parallel study where the spleens were collected fromwildtype mice at d8 post-infection and analyzed for viral burden. Thesedata demonstrate that poly-U/UC RNA is a potent antiviral therapy.

FIGS. 14A-14B illustrate the experimental approach (FIG. 14A) and theresulting survival data (FIG. 14B) demonstrating that polyU/UC RNAenhances vaccine-mediated protection of mice from West Nile virus (WNV)challenge. C57Bl6 mice were vaccinated with 100 mg polyU/UC RNA alone,0.25 mg UV-inactivated WNV +PBS, or 0.25 mg UV-inactivated WNV mixedwith 100 mg polyU/UC RNA or xRNA control. Mice were challenged 3 weekslater with 1000 pfu WNV (Tx-02) administered via subcutaneous injectionof the left footpad. Mice were monitored daily for body weight andclinical scores. Mice that lose greater than or equal to 20% body weightor that exhibit clinical symptoms greater than 5 were euthanizedaccording to UW IACUC protocols. The graph in FIG. 14B shows percentsurvival/morbidity curve for mice over a course of 21 dayspost-infection (starting at Day 0, as indicated in FIG. 14A).Differences shown between vaccine +poly-U/UC and other lines aresignificant (P<0.03). This demonstrates that poly-U/UC RNA is aneffective vaccine adjuvant for enhancement of protection by RNA virusvaccines.

DETAILED DESCRIPTION

Viral infection of vertebrate host cells triggers the innate immuneresponse through non-self recognition of pathogen associated molecularpatterns (PAMPs), such as PAMPs in viral nucleic acid. Accurate PAMPdiscrimination is essential to avoid self-recognition that can generateautoimmunity, and therefore should be defined by the presence ofmultiple motifs in a PAMP that mark it as non-self. Furthermore, thedegree of innate immune response should be properly controlled to avoidcostly damage to healthy host tissue in an excessive immune response.

Hepatitis C virus (HCV) RNA is recognized as non-self by RIG-I throughthe presence of a 5′-triphosphate (5′-ppp) on the viral RNA inassociation with an untranslated 3′ poly-U/UC tract. As described inmore detail below in Example 1, the inventors defined the specific HCVPAMP and the criteria for RIG-I non-self discrimination of HCV byexamining the RNA structure-function attributes that impart PAMPfunction to the poly-U/UC tract. It was determined that a poly-uracil(U) “core” of this sequence tract was essential for RIG-I activation,and that interspersed ribocytosine nucleotides between poly-U sequencesin the general RNA 3′ region helped to achieve optimal RIG-I signalinduction. 5′-ppp-poly-U/UC RNA variants that stimulated strong RIG-Iactivation efficiently bound purified RIG-I protein in vitro, and RNAinteraction with both the repressor domain and helicase domain of RIG-Iwas required to activate signaling. When appended to 5′-ppp RNA thatlacks PAMP activity, the poly-U/UC U-core sequence conferred non-selfrecognition of the RNA and innate immune signaling by RIG-I.Importantly, HCV poly-U/UC RNA variants that strongly activated RIG-Isignaling triggered potent anti-HCV responses in vitro and hepaticinnate immune responses in vivo using a mouse model of PAMP signaling.These data indicate a multi-motif PAMP signature of non-self recognitionby RIG-I that incorporates a 5′-ppp with varying poly-uracil sequencecomposition and length. This HCV PAMP motif drives potent RIG-Isignaling to induce the innate immune response to infection. Thesestudies define a basis of non-self discrimination by RIG-I and offerinsights into the antiviral therapeutic potential of targeted RIG-Isignaling activation.

Importantly, it was also found that the strength of RIG-I interactionand subsequent signaling varied with the length of the poly-uracil core.Accordingly, by the incorporation of poly-uracil cores of variablelengths, and the appropriate interspersing of non-uracil residues in theremainder of the general 3′-polyU/UC region, a PAMP can be rationallydesigned to elicit an innate immune response of appropriate intensity toprovide an effective response while avoiding undue damage to hosttissue, such as septic shock.

In Example 2, the inventors further demonstrate the utility of theHCV-derived poly-U/UC PAMP RNA constructs to elicit effective cellularresponses to infection by a variety of virus types. As described in moredetail in Example 2, it was determined that administration of poly-U/UCPAMP RNA constructs with U-core sequences over 8 nucleotides can lead toinnate response signaling and reduction of viral load in vitro for avariety of viruses infections. In Example 3, the inventors establishedthat poly-U/UC PAMP RNA constructs induced innate immune responsesignaling in vivo and led to reduced viral burden and increasedsurvival. In Example 4, the inventors established that co-administrationof poly-U/UC PAMP RNA constructs with an attenuated/killed West Nilevirus (WNV) vaccine significantly enhanced the protective capacity ofthe vaccine against infection by infective WNV.

In accordance with the above discovery, in one aspect, the presentdisclosure provides a synthetic nucleic acid pathogen-associatedmolecular pattern (PAMP).

As used herein, the term “nucleic acid” refers to a polymer of monomerunits or “residues”. The monomer subunits, or residues, of the nucleicacids each contain a nitrogenous base (i.e., nucleobase) a five-carbonsugar, and a phosphate group. The identity of each residue is typicallyindicated herein with reference to the identity of the nucleobase (ornitrogenous base) structure of each residue. Canonical nucleobasesinclude adenine (A), guanine (G), thymine (T), uracil (U) (in RNAinstead of thymine (T) residues) and cytosine (C). However, the nucleicacids of the present disclosure can include any modified nucleobase,nucleobase analogs, and/or non-canonical nucleobase, as are well-knownin the art. Modifications to the nucleic acid monomers, or residues,encompass any chemical change in the structure of the nucleic acidmonomer, or residue, that results in a noncanonical subunit structure.Such chemical changes can result from, for example, epigeneticmodifications (such as to genomic DNA or RNA), or damage resulting fromradiation, chemical, or other means. Illustrative and nonlimitingexamples of noncanonical subunits, which can result from a modification,include uracil (for DNA), 5-methylcytosine, 5-hydroxymethylcytosine,5-formethylcytosine, 5-carboxycytosineb-glucosyl-5-hydroxy-methylcytosine, 8-oxoguanine, 2-amino-adenosine,2-amino-deoxyadenosine, 2-thiothymidine, pyrrolo-pyrimidine,2-thiocytidine, or an abasic lesion. An abasic lesion is a locationalong the deoxyribose backbone but lacking a base. Known analogs ofnatural nucleotides hybridize to nucleic acids in a manner similar tonaturally occurring nucleotides, such as peptide nucleic acids (PNAs)and phosphorothioate DNA.

The five-carbon sugar to which the nucleobases are attached can varydepending on the type of nucleic acid. For example, the sugar isdeoxyribose in DNA and is ribose in RNA. In some instances herein, thenucleic acid residues can also be referred with respect to thenucleoside structure, such as adenosine, guanosine, 5-methyluridine,uridine, and cytidine. Moreover, alternative nomenclature for thenucleoside also includes indicating a “ribo” or deoxyrobo” prefix beforethe nucleobase to infer the type of five-carbon sugar. For example,“ribocytosine” as occasionally used herein is equivalent to a cytidineresidue because it indicates the presence of a ribose sugar in the RNAmolecule at that residue. The nucleic acid polymer can be or comprise adeoxyribonucleotide (DNA) polymer, a ribonucleotide (RNA) polymer,including mRNA. The nucleic acids can also be or comprise a PNA polymer,or a combination of any of the polymer types described herein (e.g.,contain residues with different sugars).

In some embodiments, the synthetic nucleic acid PAMP is an RNAconstruct. In some of these embodiments, the synthetic nucleic acid PAMPis derived from, or reflects the sequence of, the HCV poly-U/UC regionand, thus, may be generally referred to as the poly-U/UC PAMP RNAconstruct.

As used herein, the term “synthetic,” with reference to the syntheticnucleic acid PAMP, refers to non-natural character of the nucleic acid.Such nucleic acids can be synthesized de novo using standard synthesistechniques. Alternatively, the nucleic acid PAMPs can be generated orderived from naturally occurring pathogen sequences using recombinanttechnologies, which are well-known in the art. In some embodiments, thesequence of the synthetic nucleic acid PAMP construct is not naturallyoccurring. Descriptions of illustrative approaches to generate syntheticnucleic acid PAMPs are provided in more detail below.

The synthetic nucleic acid PAMP of this aspect comprises (a) a 5′-armregion comprising a terminal triphosphate; (b) a poly-uracil core (alsoreferred to as a poly-U core); and (c) a 3′-arm region. In oneembodiment, the three regions (a, b, and c) are covalently linked in asingle nucleic acid polymer macromolecule. The covalent linkage can bedirect (without interspersed linker sequence(s)) or indirect (withinterspersed linker sequences(s)). In one embodiment, the 5′-arm regionis covalently linked to the 5′-end of the poly-U core. In one embodimentthe 3′-arm region is covalently linked to the 3′-arm region of thepoly-U core. The polymer can be single or double stranded, or can appearwith a combination of single and double stranded portions.

As described in more detail below, it is demonstrated for the first timeherein that HCV-derived RNA PAMPs with poly-uracil core sequences havethe capacity to trigger RIG-I signaling and, thus, can stimulate aninnate immune response capable of reducing viral load, yet isappropriate to avoid septic shock. Accordingly, in one embodiment, thepoly-U core comprises at least 8 contiguous uracil residues. In furtherembodiments, the comprises between 8 and 30 contiguous uracil residues,such as 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, or 30 contiguous uracil residues. In oneembodiment, the poly-U core comprises more than 8 contiguous uracilresidues. In one embodiment, the poly-U core comprises 12 or morecontiguous uracil residues. In some embodiments, the poly-U coreconsists of a plurality of contiguous uracil residues, such as 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, or 30 contiguous uracil residues.

In one embodiment, the 3′-arm region comprises a 5′-most nucleic acidresidue that is not a uracil residue. Instead, the 5′-most nucleic acidresidue of the 3′-arm region can be an adenine, guanine, or cytosineresidue, or any non-canonical residue. In one embodiment, the 5′-mostnucleic acid residue of the 3′-arm region is a cytosine residue or aguanine residue.

In one embodiment, the nucleotide composition of the 3′-arm region is atleast 40% uracil residues. In some embodiments, the 3′-arm region is atleast 45%, is at least 50%, is at least 60%, is at least 70%, is atleast 80%, or is at least 90% uracil residues. In one embodiment, the3′-arm region comprises a plurality of short stretches (for example,between about 2 and about 15 nucleotides in length) of contiguous uracilresidues with one or more cytosine residues interspersed therebetween.In one embodiment, the 3′-arm region comprises a stretch of consecutiveuracil residues that does not exceed the length of the poly-U core ofthe synthetic PAMP construct. In one embodiment, the 3′-arm region doesnot comprises a stretch of consecutive uracil residues that exceeds thelength of the poly-U core of the synthetic PAMP construct. In someembodiments, the 3′-arm region comprises at least 7 consecutive uracilresidues, such as 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, or 29, contiguous uracil residues.

At a minimum, the 5′-arm region consists of a terminal tri-phosphate(ppp) moiety. In such embodiment, the triphosphate is at the 5′-terminusof the synthetic nucleic acid PAMP and can be represented as “5′-ppp”.In a further embodiment, the terminal triphosphate is linked directly tothe 5′-end of the poly-U core sequence. In an alternative embodiment,the 5′-arm region comprises the 5′-end terminal triphosphate and one ormore additional nucleic acid residues, the sequence of which terminateswith a 3′-end. The one or more additional nucleic acid residues in the5′-arm region of this embodiment are disposed between the terminaltriphosphate and the 5′-most uracil residue of the poly-U core. Personsof ordinary skill in the art will readily appreciate that the one ormore additional nucleic acid residues in the 5′-arm region can be anynumber of nucleic acid residues and can present any sequence withoutlimitation. As is described in more detail below, the sequence of theone or more additional nucleic acid residues in the 5′-arm region doesnot affect the functionality of the synthetic PAMP. For instance, it isdescribed that the addition of a poly-U core region to a non-stimulatorynucleic acid that contains a 5′-triphosphate (such as the HCV X region)confers stimulatory properties for innate immune system signaling. Inone embodiment, the sequence of the one or more additional nucleic acidresidues in the 5′-arm region does not consist of the entire 5′-endportion of a naturally occurring HCV genome sequence that naturallyoccurs “upstream” or 5′ to the poly-U core of the poly-U/UC region forthat HCV strain. Stated differently, in this embodiment the entiresynthetic nucleic acid PAMP construct is not a naturally occurring HCVgenome, complete with the 5′ triphosphate, the entire coding region, andthe untranslated 3′ poly-U/UC region. Accordingly, in this embodiment,the 5′-arm region, the one or more nucleic acid residues of the 5′-armregion, and the poly-uracil core do not naturally occur together in anHCV genome. However, in this embodiment, the one or more nucleic acidresidues of the 5′-arm region can comprise or consist of a subfragmentof the entire naturally occurring sequence that exists between the5′-arm region and the poly-uracil core. Alternatively, in thisembodiment, the one or more nucleic acid residues of the 5′-arm regioncan comprise sequence in addition to a portion or the entire naturallyoccurring HCV genome sequence that exists between the 5′-end and thepoly-uracil core.

The synthetic PAMP of Claim 1, wherein the synthetic PAMP is capable ofinducing retinoic acid-inducible gene I (RIG-I)-like receptor (RLR)activation. In one embodiment, the RLR is RIG-I. Persons of ordinaryskill in the art would readily be able to determine the activation of anRLR, such as by assaying the transcription of known downstreamRLR-regulated genes, as described in more detail below. For example, insome embodiments, RLR activation can be established by an increase inIFN-β or ISG54 expression. In another embodiment, RLR activation can beestablished by an increase in IRF-3 phosphorylation.

In some embodiments, the synthetic nucleic acid PAMP further comprisesand additional nucleic acid domain that encodes a functional geneproduct, such as a polypeptide or interfering RNA construct. Theadditional nucleic acid domain can be part of the 3′-arm domain, as thewhole or part of the one or more additional nucleic acid residuestherein. In another embodiment, the additional nucleic acid domain canbe disposed between any of the 5′-arm region comprising a terminaltriphosphate, the poly-uracil core, and the 3′-arm region. In someembodiments, the nucleic acid domain further comprises promoter regions,known in the art, to facilitate the general or inducible expression ofthe functional gene product in a host cell. Such gene products can beselected based on specific functionality, such as gene products that canfurther assist in the treatment or prevention of pathogen infections.

In another aspect, the present disclosure provides a pharmaceuticalcomposition comprising the synthetic nucleic acid PAMP of Claim 1.

In some embodiments, the pharmaceutical composition further comprises anattenuated, inactivated, or killed virus, bacterium, protozoan, fungus,and/or helminth. The inclusions of such attenuated, inactivated, orkilled virus, bacterium, protozoan, fungus, and/or helminth providesadditional antigens that that can be recognized by components of theadaptive immune system. For example, the additional antigen maystimulate antibody production, or when presented on MHC, may stimulate aT-cell receptor to trigger antigen-specific responses of B and T cells,respectively. With the stimulation of the adaptive immune system, andspecific memory cells that are specific to the antigen, a more long termprotection against the pathogen can be generated in addition toconferring immediate protection.

In other embodiments, the pharmaceutical composition further comprisesan antigen derived from a pathogen, such as a viral antigen, a bacterialantigen, a protozoal antigen, a fungal antigen, and/or a helminthantigen, and the like.

In some embodiments, the pharmaceutical composition comprises anyadditional known therapeutic composition, such as anti-viralcompositions, anti-biotic therapeutic, anti-fungal therapeutic,anti-protozoal therapeutic, and anti-helminth therapeutic, as are knownin the art. Co-administration of such therapeutic(s) with the syntheticnucleic acid PAMP constructs in a pharmaceutical composition can providethe advantage of triggering a multi-approach to responding to thepresence of a pathogen.

In any of the above embodiments, the virus can be any known pathogenicvirus of interest. For example, the virus can be a member of, or isderived from, the Flaviviridae, Paramyxoviridae, Hepaciviridae,Orthomyxoviridae, Bunyaviridae, Arenaviridae, Reoviridae, Retroviridae,Enteroviruses, Picornaviridae, Coronaviridae, or Noroviridae families,or the viral antigen is derived from a virus of the Flaviviridae,Paramyxoviridae, Hepaciviridae, Orthomyxoviridae, Bunyaviridae,Arenaviridae, Reoviridae, Retroviridae, Enteroviruses, Picornaviridae,Coronaviridae, or Noroviridae families. Specific examples include WestNile virus, dengue virus, Japanese encephalitis virus, vesicularstomatitis virus, hepatitis C virus, respiratory syncytial virus, yellowfever virus, influenza A virus, Lassa fever virus, Hantavirus,lymphocytic choriomenengitis virus, polio virus, parainfluenza virus,rotavirus, human immunodeficiency virus (HIV), human T-lymphotropicvirus (HTLV), enterovirus 21 and strains thereof, severe acuterespiratory syndrome (SARS) virus, Middle East respiratory syndrome(MERS) virus, corona virus, or norovirus. In some embodiments, theattenuated, inactivated, or killed virus, is derived from any of theabove classifications, for example, by multiple passages through cellculture or modification through any molecular technique known in theart, such as recombinant technologies.

As will be appreciated by persons of skill in the art, thepharmaceutical composition can comprise additional components to enhancefunctionality or stability of the pharmaceutical composition for use inany relevant and appropriate application. For example, thepharmaceutical composition can further comprise one or more of thefollowing: an adjuvant (e.g., a vaccine delivery system and/orimmunostimulatory compound), stabilizer, buffer, surfactant, controlledrelease component, salt, and/or a preservative, depending on theintended formulation for administration, as would be readily determinedby persons of skill in the art.

The pharmaceutical composition described herein can include an adjuvant.As used herein, the term “adjuvant” can be broadly separated into twoclasses, based on the principal mechanisms of action: carrier/deliverysystems and immunostimulatory compounds.

A variety of carrier or therapeutic delivery systems are known and canbe applied to the pharmaceutical composition. Delivery systems caninclude particle formulations, such as emulsions, microparticles,immune-stimulating complexes (ISCOMs), nanoparticles, which can be, forexample, particles and/or matrices, and liposomes, and the like, whichare advantageous for the delivery of antigens.

In addition, or alternatively, an adjuvant is provided to generate asignal to the immune system so that it facilitates a response to theantigen, wherein the antigen drives the specificity of the response tothe pathogen. Such “immunostimulatory” compound adjuvants are sometimesderived from pathogens and can represent pathogen associated molecularpatterns (PAMP), e.g., lipopolysaccharides (LPS), monophosphoryl lipid(MPL), or CpG-containing DNA, which activate cells of the innate immunesystem. However, it is noted that the optional adjuvant referred toherein refers to a component that is in addition to, and distinct from,the novel synthetic nucleic acid PAMP that is provided in thisdisclosure. Organic adjuvants can also include toxins produced bypathogens, such as cholera toxin.

Additionally, preferred inorganic adjuvants include aluminum salts(alum) such as aluminum phosphate, amorphous aluminum hydroxyphosphatesulfate, and aluminum hydroxide, which are commonly used in humanvaccines and are easily adapted to new vaccine technologies.

Typically, the same adjuvant or mixture of adjuvants is present in eachdose of the pharmaceutical composition. Optionally, however, an adjuvantcan be administered with the first dose of the pharmaceuticalcomposition and not with subsequent doses (i.e., booster shots).Alternatively, a strong adjuvant can be administered with the first doseof the pharmaceutical composition and a weaker adjuvant or lower dose ofthe strong adjuvant can be administered with subsequent doses. Theadjuvant can also be selected according to the relative efficacy of theadjuvant in consideration of the selected strength of the syntheticnucleic acid PAMP, which can vary depending on the length of the poly-Ucore.

In addition to the additional antigens and the adjuvants describedabove, the pharmaceutical composition formulation can include one ormore additional components, such as a stabilizer, buffer, surfactant,controlled release component, salt, and/or preservative, as arewell-known in the art.

In another aspect, the present disclosure provides a method of inducingRLR signaling in a cell. The method comprises contacting the cell withan effective amount of the synthetic nucleic acid PAMP described herein.In some embodiments, the RLR is RIG-I. The method can be performed invitro, ex vivo, or in vivo. In some embodiments, the method is repeatedone or more times. In some embodiments, the induction of RLR signalingis detectable by an increase in IFN-β levels, an increase in ISG54levels, an increase in IRF3 phosphorylation, or an increase in theexpression of any other interferon stimulated gene regulated by RLRssuch as RIG-I.

In another aspect, the present disclosure provides a method of treatinga condition in a subject treatable by inducing RLR signaling. Suchconditions include any infection by a pathogen that is treatable byinduction or enhancement of an innate immune response. Such pathogensare well-known, and include viruses, bacteria, protozoa, fungi, andhelminth parasites.

As used herein, the term “treating” with reference to any condition,disease or infection includes preventing (e.g., a prophylactic use) thecondition, disease or infection. In this context, the term treatingrefers to preventing or suppressing the infection of colonization of apathogen. Additionally, the term “treating” refers to a therapeutic use,such as addressing an infection that has already started. In oneembodiment, the term “treating” refers to curing the infection to apoint where no active pathogens remain in the host. In anotherembodiment, the term “treating” also encompasses slowing or inhibitingthe spread of the infection within the body, such as slowing orinhibiting the replication rate of the pathogen. The term alsoencompasses reducing the pathogenic burden in a cell (or host tissue orbody). The term also encompasses accelerating the rate of clearance ofthe pathogen relative to the time period required by the host'sendogenous immune response to clear the pathogen without administrationof the synthetic nucleic acid PAMP. Finally, the term “treating” alsoencompasses ameliorating the symptoms caused by pathogenic infection.

In yet another aspect, the present disclosure also provides a method ofinducing an innate immune response in a subject. The method comprisesadministering to the subject an effective amount of the pharmaceuticalcomposition as described herein. As described, the pharmaceuticalcomposition can be formulated for any appropriate route ofadministration. The method comprises administering an effective amountof the pharmaceutical composition to the subject in need thereof. Thepathogenic burden can be monitored by any known technique for detectionand quantification of the pathogen. As described above, thepharmaceutical composition can comprise additional components thataddress the disease or condition, such as pathogenic antigens to furtherstimulate the innate and/or adaptive immune responses, additionaltherapeutic agents, and the like.

In yet another aspect, the present disclosure also provides a method oftreating a viral, bacterial, protozoal, fungal, and/or helminthinfection in a subject. The method comprises administering to thesubject an effective dose of the pharmaceutical composition describedherein. As with other aspects of the disclosure, the pharmaceuticalcomposition can comprise other therapeutic components, such as pathogenagents, attenuated, killed, or inactivated pathogens, therapeuticagents, such as antibiotics and the like), additional adjuvants, and thelike.

In preferred embodiments of any of the methods described herein, theadministration or application of pharmaceutical composition comprisingthe synthetic nucleic acid PAMP does not induce septic shock to thesubject.

In any of the above methods, the pharmaceutical compositions can beappropriately formulated for preferred routes of administrationaccording to known methods. The pharmaceutical composition can beformulated for delivery by any route of systemic administration (e.g.,intramuscular, intradermal, subcutaneous, subdermal, transdermal,intravenous, intraperitoneal, intracranial, intranasal, mucosal, anal,vaginal, oral, or buccal route, or they can be inhaled). Certain routesof administration are particularly appropriate for pharmaceuticalcompositions intended to induce, at least, an innate immune response. Inparticular, transdermal administration, intramuscular, subcutaneous, andintravenous administrations are particularly appropriate.

The formulations suitable for introduction of the pharmaceuticalcompositions vary according to route of administration. Formulationssuitable for parenteral administration, such as, for example, byintraarticular (in the joints), intravenous, intramuscular, intradermal,intraperitoneal, intranasal, and subcutaneous routes, include aqueousand non-aqueous, isotonic sterile injection solutions, which can containantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The formulations can be presented in unit-dose or multi-dose sealedcontainers, such as ampoules and vials.

As will be readily appreciated, the amount of nucleic acid PAMP in theadministered formulation will vary depending upon the design of thenucleic acid PAMP (e.g., the “strength” as determined by the length ofthe poly-U core), as well as the inclusion and identity of anyadditional adjuvants or antigens.

The above methods can also comprise one or more administrations to thesubject (or to the culture of cells). The doses of pharmaceuticalcomposition can be the same or different for each dose in theadministration regime, as can be readily determined by skilled personsin the art.

In any of the above methods, the administration or application ofsynthetic nucleic acid PAMP can be in conjunction or in association withthe administration of a vaccine to stimulate a response against apathogen. As described, in some instances, the pharmaceuticalcomposition containing the synthetic nucleic acid PAMP can furthercomprise a pathogen antigen or even the entire killed, inactivated, orattenuated pathogen to stimulate an immune response and/or generateimmunological memory against the pathogen. In another embodiment, thesynthetic nucleic acid PAMP can be administered separately, but incoordination with a vaccine to achieve the same effects. The inducementof the innate immune response by the synthetic nucleic acid PAMP canprovide enhanced protection against the pathogen as compared to theprotection provided by the vaccine alone. Without being bound by anyparticular theory, the added protection likely results from the directanti-pathogenic effects of the innate immune response, in addition tothe enhanced stimulation of the adaptive immune response mechanismsthrough cross-signaling provided by the stimulated cells of the innateimmune system. An example of this effect is described in more detail inExample 4.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.”

Following long-standing patent law, the words “a” and “an,” when used inconjunction with the word “comprising” in the claims or specification,denotes one or more, unless specifically noted.

The use of the term “about” is intended to include a slight variation,such as 10%, above and below the stated value.

Unless the context clearly requires otherwise, throughout thedescription and the claims, the words “comprise,” “comprising,” and thelike are to be construed in an inclusive sense as opposed to anexclusive or exhaustive sense; that is to say, in the sense of“including, but not limited to.” Words using the singular or pluralnumber also include the plural and singular number, respectively.Additionally, the words “herein,” “above,” and “below,” and words ofsimilar import, when used in this application, shall refer to thisapplication as a whole and not to any particular portions of theapplication.

Disclosed are materials, compositions, and components that can be usedfor, can be used in conjunction with, can be used in preparation for, orare products of the disclosed methods and compositions. It is understoodthat, when combinations, subsets, interactions, groups, etc., of thesematerials are disclosed, each of various individual and collectivecombinations is specifically contemplated, even though specificreference to each and every single combination and permutation of thesecompounds may not be explicitly disclosed. This concept applies to allaspects of this disclosure including, but not limited to, steps in thedescribed methods. Thus, specific elements of any foregoing embodimentscan be combined or substituted for elements in other embodiments. Forexample, if there are a variety of additional steps that can beperformed, it is understood that each of these additional steps can beperformed with any specific method steps or combination of method stepsof the disclosed methods, and that each such combination or subset ofcombinations is specifically contemplated and should be considereddisclosed. Additionally, it is understood that the embodiments describedherein can be implemented using any suitable material such as thosedescribed elsewhere herein or as known in the art.

General texts which describe molecular biological techniques usefulherein, including the use of vectors, promoters and many other relevanttopics, include Berger and Kimmel, Guide to Molecular CloningTechniques, Methods in Enzymology Volume 152, 1987 (Academic Press,Inc., San Diego, Calif.) (“Berger”); Sambrook et al., Molecular Cloning:A Laboratory Manual, 2d ed., Vol. 1-3, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y., 1989 (“Sambrook”); and Current Protocols inMolecular Biology, F. M. Ausubel et al., eds., Current Protocols, ajoint venture between Greene Publishing Associates, Inc. and John Wiley& Sons, Inc., (supplemented through 1999) (“Ausubel”). These and anyother publications cited herein and the subject matter for which theyare cited are hereby specifically incorporated by reference in theirentireties.

EXAMPLES Example 1

Summary

Pathogen recognition receptors (PRRs) are critical components of theinnate immune response to viral pathogens, and function in the host torecognize pathogen-associated molecular patterns (PAMPs) in pathogenproteins or nucleic acids. Retinoic acid-inducible gene I (RIG-I) is acytoplasmic PRR that senses viral RNA inside an infected cell. Forexample, RIG-I recognizes hepatitis C virus (HCV) RNA as non-selfthrough the presence of both a 5′-triphosphate (5′-ppp) and a 3′poly-U/UC tract within the viral RNA. This Example describes theexamination of the RNA structure-function attributes that define the HCVpoly-U/UC tract as non-self to RIG-I, including nucleotide composition.It was found that a 34 nucleotide poly-uridine “core” (U-core) withinthe HCV poly-U/UC tract RNA stimulated non-self recognition by RIG-I,and interspersed ribocytosine nucleotides were also important to induceoptimal RIG-I signaling. Furthermore, constructs with poly-uridine coresas short as 8 nucleotides stimulated RIG-I signaling. RIG-I/RNA bindingstudies revealed that RIG-I formed weaker interactions with HCV RNAslacking poly-U sequences, and RNA interaction with multiple domains ofRIG-I was required to activate signaling. Finally, RIG-I recognition ofthe U-core within the poly-U/UC tract activated anti-HCV responses invitro and hepatic innate immune responses in vivo. These studiesidentify long poly-uridine sequences with interspersed ribocytosines asan HCV PAMP motif that drives optimal RIG-I signaling. The results ofthese studies were also published in Schnell, G., et al., “UridineComposition of the Polu-U/UC Tract of HCV RNA Defines Non-SelfRecognition by RIG-I,” PLOS Pathogens 8(8):e1002839 (2012), incorporatedherein by reference in its entirety.

Introduction

Mammalian cells respond to pathogens, such as in acute virus infections,through the actions of host pathogen recognition receptors (PRRs) thatrecognize viral pathogen-associated molecular patterns (PAMPs). TheRIG-I-like receptors (RLRs) are cytoplasmic RNA helicases that functionas PRRs for the recognition of RNA virus infection. The RLRs includeRIG-I (retinoic acid-inducible gene I), MDA5 (melanomadifferentiation-associated gene 5), and LGP2 (laboratory of genetics andphysiology 2). Whereas RIG-I and MDA5 encode tandem amino-terminalcaspase activation and recruitment domains (CARDs), LGP2 lacks CARDs andis thought to play a regulatory role in signaling initiated by RIG-I orMDA5. Following the recognition and binding of viral PAMP RNA, RIG-Isignals through the adaptor protein mitochondrial antiviral signaling(MAVS, also known as IPS-1/VISA/Cardif). Downstream signaling by theRLRs induces the activation of latent transcription factors, includinginterferon regulatory factor (IRF)-3 and NF-κB, leading to theproduction of type-I interferons (IFN) from the infected cell. Local IFNsecretion leads to the expression of hundreds of interferon-stimulatedgenes (ISGs) in the infected cell and surrounding tissue that mediateantiviral and immunomodulatory properties in order to restrict virusreplication and impart the onset of the immune response to infection.

The process of RIG-I signaling activation has been revealed throughstructure-function studies. In addition to the N-terminal CARDs, RIG-Ipossesses a central DExD/H box RNA helicase/ATPase domain and aC-terminal repressor domain (RI)). RIG-I recognizes and binds tospecific PAMP motifs within RNA marked by a free/exposed 5′-triphosphate(5′-ppp), including single-stranded (ss)RNA or double-stranded (ds)RNA.RIG-I binding interactions with viral RNA are mediated through multiplecontacts with the helicase domain and the C-terminal RD, the latter ofwhich binds 5′-ppp motifs with high specificity. RIG-I recognition andbinding of viral RNA relieves auto-repression and drives ATP hydrolysisand conformational rearrangements that expose the CARDs for downstreamsignaling to initiate the immune response to infection. Despite therecent advancements in RIG-I structural biology, the nature of RIG-Irecognition of sequence-specific PAMP RNA motifs remains unclear.Accurate discrimination of self from non-self by PRRs is essential toavoid immune triggering against self that leads to autoimmunity. In thissense, PRR recognition of a single PAMP motif alone, such as 5′-ppp,within viral RNA is unlikely to accurately discriminate the comparablylow abundance PAMP RNA from the high abundance host RNA. Moreover, thepresence of a single motif within host RNA that displays the PAMPsignature could induce aberrant signaling against self, whereas acombinatorial non-self signature for PRR binding and signalingactivation would serve to accurately discriminate it as a PAMP. Previousstudies have revealed that multiple parameters define an RNA PAMP forRIG-I recognition, including 5′-ppp, length (>19 nt), secondarystructure characteristics, and nucleotide sequence motifs.

RIG-I is essential for host cell recognition of a variety of RNAviruses, including hepatitis C virus (HCV). HCV is a positive-sensessRNA virus that replicates in hepatocytes and causes chronic liverdisease and liver cancer. Approximately 200 million people worldwide arepersistently infected with HCV, and infection is characterized bychronic viral replication, producing viral RNA that can trigger innateimmune responses. HCV RNA is recognized as non-self by RIG-I throughrecognition of the poly-U/UC tract located in the 3′ non-translatedregion (NTR) of the viral genomic RNA, thus defining the poly-U/UC tractas a PAMP motif of HCV. The HCV poly-U/UC tract is approximately 100nucleotides (nt) in length and is essential for virus replication andviability. While the poly-U/UC tract is conserved in 39 NTR placementwithin all genotypes and strains of HCV, it varies in the length ofpoly-uridine sequences and the positioning of ribocytosine nucleotides.RIG-I recognition of the HCV poly-U/UC tract is dependent on the 5′-pppRNA length and sequence composition, and RIG-I signaling is attenuatedin response to HCV poly-U/UC RNAs shorter than 50 nucleotides in lengthor with a reduced poly-uridine nucleotide composition compared towild-type viral RNA. Although RIG-I recognizes HCV RNA, the specific RNAsequence motifs in the HCV poly-U/UC tract that confer RIG-I recognitionare not known.

In this study, the properties of RIG-I recognition of HCV RNA wereevaluated by conducting a detailed structure-function analysis of RIG-Iand poly-U/UC RNA interactions and innate immune signaling. The resultsshow that the poly-uridine core (U-core) within the HCV poly-U/UC tractwas essential for recognition by RIG-I, indicating that RIG-I recognizeslong poly-uridine regions as non-self motifs within 5′-ppp RNA. Inaddition, the affinity of RIG-I/RNA binding interactions, and criticalcontacts between the PAMP RNA and RIG-I helicase domain, both defined anHCV RNA recognition sequence in which long poly-uridine sequences (>U17)with interspersed ribocytosines induced in vitro anti-HCV responses andhepatic innate immune responses in vivo. Furthermore, it wasdemonstrated that HCV RNA sequences in which shorter poly-uridinesequences with at least 8U core sequences stimulated some IFN-βsignaling. Thus, RIG-I recognition of the U-core within the poly-U/UCtract of the 5′-ppp HCV RNA is the trigger of innate antiviral immunityto HCV infection. Poly-uridine sequences could thereby offer a novelapplication for innate immune stimulation in vaccine vectors andantimicrobial therapeutic strategies for controlling infections.

Results

The U-core of the HCV poly-U/UC tract is required for RIG-I signalinduction.

To determine the RNA sequence elements in the HCV poly-U/UC tractrequired for non-self recognition by RIG-I, multiple poly-U/UC RNAconstructs were developed that encoded changes in distinct regionstermed the 5′arm, U-core, and 3′arm, and based on the HCV genotype 1bconsensus (Con1) poly-U/UC sequence (Table 1). Two RNA constructsencoding either the HCV Con1 3′ NTR X-region sequence alone, orincluding a 34 nt poly-uridine sequence between stem-loops 1 (SLI) and 2(SLII) of the X-region were also developed. Each RNA was generated invitro from a DNA template using the T7 RNA polymerase, which resulted inthe RNA products having a 5′-ppp and three guanine nt at the 5′ end.Previous studies demonstrated that the poly-U/UC tract, but not theX-region, of the HCV 3′ NTR was responsible for RIG-I recognition andtriggering of innate immune signaling. The ability of equal moles of5′-ppp full-length JFH1 HCV RNA or the JFH1 poly-U/UC tract to activateRIG-I signaling to the IFN-β-promoter in human hepatoma (Huh7) cellsharboring an intact RIG-I pathway were assessed. The full-length HCV RNAgenome and the poly-U/UC tract RNA from either genotype 1b (Con1) or 2a(JFH1) were able to activate signaling and drive transcription from theIFN-β-promoter (FIG. 1A), confirming that the poly-U/UC tract of the HCVRNA is a PAMP that triggers PRR signaling. Additionally, induction ofthe IFN-β-promoter by the Con1 and JFH1 poly-U/UC tract (pU/UC) RNAs inHuh7 cells (FIG. 1B) was also found to be linked with the induction ofIRF-3 phosphorylation and ISG expression (FIG. 1C). Additionally, Huh7.5cells that lack a functional RIG-I pathway failed to respond totransfected HCV RNA constructs (FIG. 1B), thus defining RIG-I-dependenceto HCV PAMP recognition and innate immune signaling.

TABLE 1HCV poly-U/UC RNA contructs developed for RIG-I binding and activation studies. RNA U- SEQ construct^(a) 5′ arm^(b) core 3′ arm ID NO: Con1 pU/UC5′GGCCAUCCUGUUUUUUUCCC(U11)C U34 CUCCUUUUUUUUUCCUCUUUUUUUCCUUUUCUUUCCUUU 1 JFH1 pU/UC 5′ACUGUUCC U43C(U14)CCCUCUUUCUUCCCUUCUCAUCUUAUUCUACUUUCUUUCUU  2 pU/UC C265′GGCCAUCCUGUUUUUUUCCC(U11)C U34CUCCUUUUUUUUUCCUCUUUUUUUCCUUUUCUUUCCUUU(C26)  3 pU/UC 3′C265′GGCCAUCCUGUUUUUUUCCC(U11)C U34 CUCCUUUUUUUUUCCCCCCCCCCCCCCCCCCCCCCCCCC 4 pU/UC C67U 5′GGCCAUCCUGUUUUUUUCCC(U11)C U34UUCCUUUUUUUUUCCUCUUUUUUUCCUUUUCUUUCCUUU  5 poly-U 1075′UUUUUUUUUUUUUUUUUUUU(U11)U U34 UUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUU 6 Δcore 5′GGCCAUCCUGUUUUUUUCCC(U11)C ---CUCCUUUUUUUUUCCUCUUUUUUUCCUUUUCUUUCCUUU  7 U8core5′GGCCAUCCUGUUUUUUUCCC(U11)C U8CUCCUUUUUUUUUCCUCUUUUUUUCCUUUUCUUUCCUUU(C26)  8 U17core r5′GGCCAUCCUGUUUUUUUCCC(U11)C U17CUCCUUUUUUUUUCCUCUUUUUUUCCUUUUCUUUCCUUU(C17)  9 pU/UC 625′GGCCAUCCUG--------------- U34 CUCCUUUUUUUUUCCUCU---------------------10 5′C 5′CCCCCCCCCC--------------- U34CUCCUUUUUUUUUCCUCU--------------------- 11 3′C5′GGCCAUCCUG--------------- U34 CCCCCCCCCCCCCCCCCC---------------------12 poly-U 62 5′UUUUUUUUUU--------------- U34UUUUUUUUUUUUUUUUUU--------------------- 13 poly-U 62-C5′UUUUUUUUUU--------------- U34 CUUUUUUUUUUUUUUUUU---------------------14 U/C1 5′UUCCUUCCUU--------------- U34CUCCUUUUUUUUUCCUCU--------------------- 15 U/C25′UUUUUUUUUG--------------- U34 CUCCUUUUUUUUUCCUCU---------------------16 U/C3 5′UUUUUUUUUG--------------- U34CUUUUUUUUUUUUCCUUU--------------------- 17 U/C45′GGUUUUCCUU--------------- U34 CUUUUUUUUUUUUUUUUU---------------------18 U/C5 5′GGCCAUCCUG--------------- U10C(U10)C(U10)CUCUCCUUUUUUUUUCCUCU------- 19 U/C65′GGCCAUCCUG--------------- U15 C(C15)CUUCUCCUUUUUUUUUCCUCU------------20 U/C7 5′GGCCAUCCUG--------------- U10CCC(U10)CCC(U8)CUCCUUUUUUUUUCCUCU------ 21 U/C85′GGCCAUCCUG--------------- U18 CCCCCC(U10)CUCCUUUUUUUUUCCUCU----------22 ^(a)The X-region RNA construct has the sequence5′-GGUGGCUCCAUCUUAGCCCUAGUCACGGCUAGCUGUGAAAGGUCCGUGAGCCGCUUGACUGCAGAGAGUGCUGAUACUGGCCUCUCUGCAGAUCAAGU-3′(SEQ ID NO: 23). The X-region-U34 RNA construct has the sequence5′-GGGUGGCUCCAUCUUAGCCCUAGUCACGGCUAGCUGUGAAAGGUCCGUGAGC(U34)CGCUUGACUGCAGAGAGUGCUGAUACUGGCCUCUCU-GCAGAUCAAGU-3′(SEQ ID NO: 24). ^(b)All RNAs include a 5′-ppp and three guaninenucleotides at the 5′ end of the RNA. Dashes indicate nucleotidedeletions, and underlined nucleotides show changes from the HCV Con1poly-U/UC sequence. Long homo-polymeric nucleotide sequences areindicated in parentheses with the nucleotide designation followed by thenumber of nucleotides in the sequence.

Huh7 and Huh7.5 cell responses to HCV poly-U/UC tract RNA constructderivatives were further evaluated (Table 1 and FIG. 1). Neither theC67U nt substitution (pU/UC C67U construct) nor the addition of 26ribocytosine nucleotides to the 3′end of the RNA (pU/UC C26)significantly changed signaling compared to wild-type poly-U/UC RNA inHuh7 cells. However, despite the presence of a 5′-ppp, deletion of theU-core from the pU/UC tract (Δcore) ablated the induction of IRF-3phosphorylation and signaling to the IFN-β-promoter, thus preventing ISGexpression (FIG. 1B and FIGURE IC). While replacement of the U-core with8 uridine nts (U8core) did not result in detectable IRF-3phophorylation, a 17 uridine nt core RNA (U17core) triggered low levelsof IFN-β signaling activity and IRF-3 phosphorylation. These dataindicate that in addition to a 5′-ppp, a U-core length of over 8 uridinenucleotides may be sufficient to stimulate some RIG-I based signaling.In addition, while the 5′-ppp HCV X-region RNA did not trigger RIG-Isignaling, it was found that insertion of a 34 nt poly-U sequence (U34)into the X-region RNA between SLI and SLII (X-region-U34) resulted inits recognition as a PAMP to stimulate IRF-3 phosphorylation,IFN-β-promoter activity, and ISG expression (FIG. 1B and FIGURE IC).Other RNA constructs with sequence changes in the U-core region (U/C5,U/C7) exhibited a significantly decreased capacity to stimulateinduction of the IFN-β-promoter (P<0.01 and P<0.001 respectively,two-tailed Student's t-test) compared to either the wild-type Con1 pU/UCRNA or the truncated pU/UC 62 RNA (encoding the wild-type U-core). Takentogether, these results demonstrate that the U-core of the HCV poly-U/UCtract exceeding 8 uridine residues is required for non-self recognitionby RIG-I and subsequent activation of the innate immune response.

Analysis of specific poly-U/UC tract constructs also revealed thatinterspersed ribocytosine nucleotides between the poly-U sequences inthe RNA were necessary to achieve optimal RIG-I signal induction. It wasnoted that substitution of the last 26 nucleotides in the 3′arm of thepoly-U/UC tract to ribocytosines (pU/UC 3′C26) resulted in a significantdecrease in IFN-β-promoter induction compared to the wild-type Con1pU/UC sequence (FIG. 1B; P=0.0047, two-tailed Student's t-test). Thus,although the U-core is required for poly-U/UC recognition by RIG-I, theuridine/ribocytosine sequences located in the 3′arm play a role in PAMPrecognition. Therefore, a set of truncated poly-U/UC constructs wasdeveloped with C to U substitutions and their PAMP activity wascompared. Activity was defined as signaling of IFN-β-promoter induction,with PAMP activity driven by the equivalent-length pU/UC 62 construct.The 5′C, 3′C, U/C1, U/C2, U/C3, and U/C4 RNA constructs each exhibitedsimilar PAMP activity as the pU/UC 62 RNA to induce the IFN-β-promoter(FIG. 1B). Removal of all ribocytosine nucleotides revealed a propertyof length-dependent, U-specific PAMP activity of which the shorterpoly-U 62 RNA induced significantly less signaling to the IFN-β-promotercompared to the longer poly-U 107 RNA in Huh7 cells (FIG. 1B; P=0.0004,two-tailed Student's t-test). However, this effect of RNA length wasovercome by inserting a single ribocytosine nucleotide into the poly-U62 RNA, wherein the poly-U 62-C RNA was able to drive IRF-3phosphorylation, IFN-β-promoter induction, and ISG expression asefficiently as the wild-type Con1 pU/UC RNA (FIG. 1B and FIGURE IC).Thus, poly-U length and ribocytosine content impact RIG-I recognitionand PAMP activity of the HCV poly-U/UC tract.

RNA binding interactions determine RIG-I signaling activation.

To examine the binding interactions between HCV poly-U/UC RNA and RIG-Ithat impart PAMP activity, an electrophoretic mobility gel-shift assay(EMSA) was conducted with purified recombinant RIG-I protein and 10 pmolof each poly-U/UC RNA construct (FIG. 2A). Comprehensive EMSA analyseswere conducted on nine RNA constructs to generate saturation-bindingcurves (FIG. 2B). The largest differences in RIG-I binding between thevarious poly-U/UC RNA constructs were detected at lower RIG-Iconcentrations (0-10 pmol), whereas increasing saturation of RIG-I/RNAbinding reactions were observed for most RNA constructs atconcentrations of RIG-I that exceeded 10 pmol. In order to compare theassociation differences between the RNA constructs and RIG-I, the pmoleffective concentration (EC) of RIG-I required to shift 10% (EC₁₀), 50%(EC₅₀), or 90% (EC₉₀) of each RNA as determined by EMSA was calculated(FIG. 2C). The EC values for the X-region, X-region-U34, Con1 pU/UC,JFH1 pU/UC, Δcore, U8core, U17core, and poly-U 62-C RNAs were calculatedusing the graphical equation generated from the comprehensive EMSAanalyses (graphically depicted in FIG. 2B). The remaining RNA constructshad more limited EMSA analyses; therefore, only the general ranges forthe EC values to between 0-10 pmol RIG-I were able to be calculated.

As shown in FIG. 2C, the EC₁₀ values for the various poly-U/UC RNAconstructs ranged from 0.65-3.04 pmol RIG-I, and EC₅₀ values ranged from2.28-7.31 pmol RIG-I. Noting that all RNAs included a 5′-ppp, it wasfound that the X-region RNA did not bind to RIG-I in the range of 0-10pmol of protein, while the X-region-U34 RNA had an EC₁₀ value of 1.42pmol RIG-I and >50% of the RNA bound to 8 pmol of RIG-I, demonstratingthat the inserted U34 sequence promoted stable binding interactionsbetween RIG-I and the X-region-U34 RNA. In addition, it was found thatEC₁₀ values were significantly larger (P=0.0005, two-tailed Student'st-test) for RNAs that did not signal (Δcore) versus RNAs thatdemonstrated PAMP activity and signaled to the IFN-β-promoter(X-region-U34, Con1 pU/UC, JFH1 pU/UC, U17core, and poly-U 62-C). Thenon-signaling Δcore RNA contain large deletions in the U-core region,while the RNAs that induced RIG-I signaling all contained poly-Usequences >8 nt in length. These data reveal that RIG-I forms only weakinteractions with 5′-ppp RNAs lacking poly-U sequences, and demonstratethat the poly-U core of the HCV pU/UC tract over 8 nucleotides long islikely required to confer stable RIG-I/RNA binding interactions. Therewas not a significant difference in the EC₅₀ or EC₉₀ values between thesignaling and non-signaling RNAs, indicating that differences in RNAaffinity may play an important role in determining RIG-I signalingactivation at lower, more physiologically relevant concentrations ofRIG-I.

RIG-I/RNA affinity among RNAs denoted as non-signaling/low signaling(Δcore, U8core), medium signaling (X-region-U34, U17core), and highsignaling (Con1 pU/UC, JFH1 pU/UC, poly-U 62-C) were also examined basedon their ability to induce different levels of signaling to the IFN-βpromoter. There was a statistically significant increase in the EC₁₀(P=0.0025, two-tailed Student's t-test) and EC₅₀ (P=0.004) valuesmeasured for no/low signaling RNAs compared to high signaling RNAs, thusdemonstrating that the Δcore and U8core RNAs (lacking a poly-U core)form significantly weaker interactions with RIG-I than those RNAs withstronger PAMP activity. A significant difference was also detected whencomparing the EC₁₀ values measured for no/low signaling compared tomedium signaling RNAs (P=0.0263). In general, the EC₉₀ for all RNAconstructs was >10 pmol RIG-I regardless of whether or not the RNA coulddrive RIG-I activation. However, there was still a significantdifference in the EC₉₀ values between RNAs that did not signal (Δcore,U8core) and the RNAs that induced high signaling to the IFN-β-promoter(Con1 pU/UC, JFH1 pU/UC, poly-U 62-C; P=0.0208 using a two-tailedStudent's t-test). Thus, these data link HCV RNA sequences containingpoly-U motifs with stronger RIG-I binding and enhanced signaling for anoverall potent PAMP activity. Taken together, the data indicate that thestrength of RIG-I/RNA binding interactions defines an HCV RNArecognition sequence in which poly-uridine sequences greater than 8uridine nucleotides with interspersed ribocytosines drive RIG-I bindingand signaling to induce the innate immune response.

RNA interactions with the RIG-I RD and helicase domain are important forPAMP signaling.

RIG-I is maintained in an auto-repressed conformation in uninfectedcells where the CARDs interact with either the C-terminal repressordomain (RD), the helicase insertion domain (Hel2i), or both domains.Crystal structure studies of RIG-I bound to RNA have revealed that theRD interacts with the 5′-ppp terminus of the RNA, which brings RNAstructures in close contact with the helicase domains to allow for morespecific RIG-I/RNA interactions. To further assess RNA interactions withRIG-I, and to determine how HCV PAMP RNA interacts with the helicasedomain and RD of RIG-I, limited-trypsin proteolysis of RIG-I/RNAcomplexes were conducted for nine RNA constructs selected to represent arange of PAMP activity (FIG. 3). RIG-I is highly sensitive to trypsinproteolysis in the absence of PAMP RNA; however, upon binding to an RNAligand RIG-I undergoes conformational changes that protect the RD andother RNA-bound domains from trypsin digestion.

It was observed that all RNA constructs were able to bind and protectthe RIG-I RD, albeit variably, from trypsin proteolysis in adose-dependent manner (FIG. 3A). While no binding between RIG-I and theX-region RNA was previously observed in the EMSA analysis, the trypsinproteolysis assay revealed limited protection of the RIG-I RD by theX-region RNA. These differences in X-region binding likely reflect thehigher amount of RIG-I required for protein visualization in thelimited-trypsin proteolysis assay, and indicate that some degree ofbackground binding occurs between RIG-I and ligand RNA in this assay.There was not a significant difference in the RD band intensity whencomparing RIG-I protection from 1 pmol of non-signaling (X-region,Δcore) versus signaling RNAs, likely reflecting binding supported by the5′-ppp on all of the RNAs (FIG. 3B). However, a dose-dependentprotection of an approximately 78 kDa proteolytic product was detectedrepresenting a large portion of the RIG-I helicase domain and CARDs(FIG. 3A). This product was only detected for RNA constructs thatexhibited PAMP activity to induce signaling to the IFN-β-promoter(X-region-U34, Con1 pU/UC, JFH1 pU/UC, U17core, poly-U 62, poly-U 62-C).In addition, there was a statistically significant difference in thehelicase+CARDs band intensity when comparing protease protection ofRIG-I resulting from 1 pmol of non-signaling versus signaling RNA (FIG.3B; P=0.0147, two-tailed Student's t-test). These observations suggestthat in addition to RD interactions, RNA interactions with the helicasedomain may be required for induction of RIG-I signaling. No significantdifference was detected in RIG-I ATPase activity when bound to thevarious RNA constructs, but the lowest ATPase activity was observed forRIG-I bound to the X-region RNA (FIG. 3C). Taken together, these resultsindicate that RNA binding interactions with the RIG-I RD are mediated bythe 5′-ppp, and specific interactions between the helicase domain andpoly-uridine tract of the HCV RNA are required for PAMP activity and toinitiate RIG-I signaling.

HCV poly-U/UC RNA variants trigger differential anti-HCV and hepaticinnate immune responses.

To determine how the poly-U core sequence imposes PAMP activity thatinitiates RIG-I signaling of innate immunity, HCV production wasmeasured in Huh7 cells that were first transfected with poly-U/UC RNAconstructs to stimulate RIG-I signaling (FIG. 4A; No RNA, X-region,X-region-U34, Con1 pU/UC, and Δcore). 12 hours following RNAtransfection, the cells were infected with HCV, and virus production wasthen assessed 48 hours later. It was observed that X-region RNA did notstimulate cellular suppression of HCV infection, whereas the Con1 pU/UCRNA stimulated a potent innate immune response that significantlysuppressed HCV infection as compared to non-transfected control cultures(FIG. 4A). These analyses were applied to a non-parametric correlationtest to reveal an inverse correlation between HCV production titer(ffu/ml) in the transfected cells and PAMP activity of the differentRNAs as measured by the IFN-β-promoter fold index in Huh7 cells(Spearman r=−0.97; two-tailed P-value=0.0167). Overall, RNA constructsthat induced higher levels of IRF-3 phosphorylation and IFN-β-promoteractivity (X-region-U34, Con1 pU/UC; FIG. 1) were more effective ininducing RIG-I signaling to suppress HCV infection.

To determine if the differential PAMP activity of HCV RNA constructsimposed different levels of hepatic innate immune signaling and responsein vivo, the ability of the constructs to trigger hepatic innate immuneresponses was examined using an intraperitoneal injection model in mice.This model recapitulates RIG-I-dependent PAMP signaling of hepaticinnate immunity triggered by HCV RNA (Saito, T., et al., “InnateImmunity Induced by Composition-Dependent RIG-I Recognition of HepatitisC Virus RNA,” Nature 454:523-527 (2008)). Four poly-U/UC RNA variants(X-region, X-region-U34, Con1 pU/UC, and Δcore) were mixed with alipid-based in vivo RNA transfection reagent and injected into wild-typeC57BL/6 mice, and livers were collected 8 hrs later for assessment ofthe hepatic innate immune response. Expression of IFN-β mRNA and severalinterferon-stimulated genes (ISGs) were measured using real-timequantitative PCR (FIG. 4B). Hepatic IFN-β, CCL5, ISG15, and Ifit2 (alsoknown as ISG54) mRNA expression were found to be significantly higher inmice transfected with Con1 pU/UC RNA compared to either X-region orΔcore RNA, indicating that a U-core of the pU/UC tract over 8 uridinenucleotides long is required for PAMP activity that induces theexpression of IFN-β and other ISGs. Expression of hepatic Ifit2 mRNA wasalso higher in mice transfected with X-region-U34 RNA compared toX-region RNA, although the difference in gene expression did not reachstatistical significance, implicating the requirement of poly-U RNAsequences for activation of hepatic innate immune responses in vivo.Expression of Ifit2 also correlated with ISG54 protein expression asshown by immunohistochemical staining of mouse liver sections (FIG. 4C).ISG54 protein expression was substantially higher in mice that receivedX-region-U34 or Con1 pU/UC RNA compared to either PBS, X-region, orΔcore RNA. Taken together, these studies indicate that >8 nt poly-U coreof the HCV poly-U/UC tract is required for PAMP activity in vivo todrive RIG-I signaling of the hepatic innate immune response.

HCV Poly-U/UC Sequence Variability

Genetic variability of the poly-U/UC tract in HCV genome sequencescontaining full coverage of the 3′NTR that were available from theGenBank sequence database was examined. Non-redundant HCV poly-U/UCsequences were aligned to examine sequence variability. It was foundthat the poly-U/UC sequences varied in both length and nucleotide (nt)composition (see Table 2). In terms of length, the 5′arm ranged from8-42 nt, the U-core ranged from 12-96 nt, and the 3′arm ranged from 0-80nt. Nine pU/UC sequences contained a U-core with fewer than 20 uridinenucleotides, suggesting that these genomes likely have low RIG-Isignaling activity. In general, HCV pU/UC genotype 1 sequences containedfewer purine nucleotides than genotype 2 sequences. Within the 5′arm ofthe pU/UC tract, genotype 1 nucleotide composition ranged from 12-44%purines compared to 16.7-50% purines in genotype 2 sequences. Within the3′arm of the pU/UC tract, 41% of the genotype 1 sequences lacked purinents (range of 0-11.6% purine composition), whereas genotype 2 sequencecomposition ranged between 5-11% purine nts in the 3′arm. We were unableto examine pU/UC sequence variability for genotypes 3-6 due to adeficiency of full-length 3′NTR sequences from those viral genotypes inthe available sequence databases. Our analysis of HCV genome sequencesreveal nt composition and length variability in the poly-U/UC tract ofthe HCV genome, and importantly within the U-core, suggesting that PAMPactivity may differ substantially between HCV genotypes and withinpatient quasispecies populations.

TABLE 2 HCV poly-U/UC sequence variability. GenBank U- SEQ Acc. Gen.^(a)5′ arm^(b) core 3′ arm^(c) ID NO: AJ238799.1b5′GGCCAUCCUGUUUUUUUCCC(U11)C U34 CUCCUUUUUUUUUCCUCUUUUUUUCCUUUUCUUUCCUUU25 AB001040.1b 5′GGCCAUUC U16 CUUUCUUCUUU 26 AB016785.1b 5′GGCCGUCCUGU18 27 AB049088.1b 5′GGCCAUUCCC U81 CUCUUCUUUUCUUUAUUCCUUCUUU 28AB049089.1b 5′GGCCGUUCC U64 CUUUUCCCCUUUUUUAUUUUUCUUUCUU 29 AB049090.1b5′GGCCAUCCUGUUUUUUUGUUUUUUC U43 CUUUUUUUCCCUUUUUUUUAUUUUAUUUUCUUUUGGU 30AB049091.1b 5′GGCCAUCCCCC U96 CCUCUUUUUUUCCUUUUCUUCUUU 31 AB049095.1b5′GGCCGUUCUG U85 CCUUUUUUUUAUUCCUCUUCU 32 AB049101.1b 5′GGCCAUCCCCUUUGU94 AUUUCUCCUUCUUUU 33 AB080299.1b 5′GGCCAUUCCU U24CUUUUUUUUUCC(U24)CCUUUUCUUUCUUCUUU 34 AB249644.1b 5′GGCCAUUUUC U14CUUCUUUCUUUUUCUUUUUCUUUUUUUCCUUCUUU 35 AF054247.1b 5′AGCCAUUUCCUG U28CUUUUUUUUUUUCUUUCCUUUCCUUCUUUUUUUCCUUUCUU 36 UUUCCCUUCUUUAAU AF139594.1b5′GGCCAUUUCCUG(U15)GG U39 CCUUUCCUUCUUUUUUUUUUUUUCCCUCUUUAU 37AF333324.1b 5′GGCCAUUUCCUG U34 CUUUUCCUUCUUUUUCCCUUUUUCUUUCUUCCUUCUUUAAU38 AF176573.1b 5′GGCCAUCCUGUG U75 AUUUCCUUUUCUU 39 AF356827.1b5′GGCCAACCUG(U26)CC U34 CCUUUUUUUCUUUUUUUUUUUUUUUUUCCUUCCUUUU 40AJ132997.1b 5′GGCCAUCCUG U16 CUUUCUUU 41 AY460204.1b 5′GGCCAUUUUUCC U23CUUUUUUUUUUCCUUUUUUUCUUUUUUUUUCUUUUCUUU 42 D85018.1b 5′GGCCAUUC U35CGUUUCUUUUUCUUCUUUUUGUUUUCUCUUCUCCUUUU 43 D85021.1b5′GGCCAUUCCCC(U14)CCGC U33 CUUUUUUUUUCC(U27)CUUUUU 44 D85022.1b5′GGCCAUCCCCC(U13)CCGC U21 CUUUUUUUUUUUCUUUUUUUUUUCC(U24)CUUUUCUUUUU 45D85516.1b 5′GGCCAUUC U16 CUUUCUUCUUU 46 D89815.1b 5′GGCCAUCCCCUUC U22CCUUUUCUUCUUU 47 EU857431.1b 5′GGCCAUCCUGUUUUUUUCCC(U11)C U29CUCCUUUUUUUUUCCUCUUUUUUUCCUUUUCUUUCCUUU 48 FN435993.1b5′GGCCGUCCUG(U19)CC U67 CUUCUUUCUUUCUU 49 GU133617.1b 5′GGCCAUUUCCUG U53C(U17)CC(U20)CUUUCCUUCUUUUUUCCUUUCUUUUCCUUC 50 CUUCUUUAAU AB520610.1a5′GGCCAUUCCUG U16 CUUUUGUUUUUUUUG(U17)CCUUUC(U15)CCUUUCUUCUUUAAU 51AF009069.1a 5′GGCCAUUUCCUG U20 CUUUCCUUCUUUUUUCCUUUCUUUUCCUUCCUUCUUUAAU52 AF009070.1a 5′GGCCAUUUCCUG U34CUUUUCCUUCUUUUUCCCUUUUUCUUUCUUCCUUCUUUAAU 53 AF009071.1a 5′GGCCAUUUCCUGU51 C(U17)CC(U20)CUUUCCUUCUUUUUUCCUUUCUUUUCCUUC 54 CUUCUUUAAUAF009072.1a 5′GGCCAUUUCCUG(U14)CCC U37CUUUCCUUCUUUUUUUUCCUUUCUUUUCCUUCCUUCUUUAAU 55 AF009074.1a 5′GGCAUCCUGU64 CUUUUCUUU 56 AF009075.1a 5′ACACUCCAUUUCUUUUUUUG U67CUUUUUCUUUCCUUUCUUUUCUGACUUCUAAUUUUCCUUCUUA 57 AF009076.1a 5′GUCCUUCUGU78 CCUUACCCUUUCCUUCUUUUCUUCCUUUUUUUUCCUUACUUU 58 AF009077.1a5′GGGUCCCCUUG U12 CUUUCCUUCUUUCCUUUCCUAAUCUUUCUUUCUU 59 AF011751.1a5′AGCCAUUUCCUG U28 CUUUUUUUUUUUCUUUCCUUUCCUUCUUUUUUUCCUUUCUUUU 60UCCCUUCUUUAAU AF271632.1a 5′GGCCAUUUCCUG(U15)G U55CCUUUCCUUUUUUUUUUUUUUUCCCUUUUUAU 61 AJ278830.1a 5′GGCCAUCCUG(U22)C U17CUUUUUUUUUCUUCUUUUUCUUUCC(U24)CUUCUUUC 62 EF621489.1a 5′GGCCAUUUCCUG U46CUUUUUCCCUCUUUUUCUUCUCUUUUUCCUUCUUUAAU 63 AB047639.2a 5′GCUAACUGUUCC U43CUUUUUUUUUUUUUUCCCUCUUUCUUCCCUUCUCAUCUUAUUCU 64 ACUUUCUUUCUU AB047640.2a5′GCUAACUGUUCC U38 C(U15)CCCUCUUUCUUCCCUUCUCAUCUUAUUCUACUUUCUUUCUU 65AB047641.2a 5′GCUAACUGUUCC(U11)C U27CCUUCUUUCUUUCUUUCUUACCUUACUUUACUUUCUUUUCU 66 AB047642.2a5′GCUAACAGUUUCUC(U13)CC(U6) U25AUUUUCUUUUCCUUUCUUUCUCACCUUACAUUACUUUCUUUCUU 67 AUUUUUA AB047643.2a5′GCUAAUUUCCUUAUUG U19 CUUUCCAUUUCCUUCCUUCUUACUUCACUUUACCUUCUUUCU 68AB047644.2a 5′GCUAACUG U77 CCUUUCCUUUCUUUCUUACCUUACUUUACAUUCUUUUCU 69AB047645.2a 5′GCUAACUGUUCC U70 CUUUCCUUCCUUUCUCACCUUCUUUUACUUCUUUCCU 70AF169002.2a 5′GCUAACUG U45 CUUUUCUUUCCUUUCCUUCUUACUCUACUUUACUUUUUCU 71AF169003.2a 5′GCUAACUGUUC U78 CUUUUCCUUCUUCUUUCUUACCUUAUUUUCCUUCUUUCUU72 AF169004.2a 5′GCUAACUG U30CUUUUUUUUUCUUUUCUUUCCUUCUUACCUUACUUUACUUUCUUUUCU 73 AF169005.2a5′GCUAACUG U81 CCUUUUUCCUUUUCCUUCUCUUUUUACCUUACUUUACUUUUCUU 74AF177036.2a 5′GCUAACUGUCCC U84CUUUUUUUCUCUUUUCCUUCUUUCUUACCUUAUUUUACUUUCUUUCCU 75 AY746460.2a5′GCUAACUGUCCCUUUUUUUUUG U30C(U18)GUUUCUUUUCCUUCUCAUUUCCUUCUUAUCUUAAUUAC 76 UUCCUUUCCU D67095.2a5′GCUAACUG U39 CCUUCUUCCUUUCCUUCUUACCUUACUUUAUUUUCUUUCCU 77 D67096.2a5′GCUAACUG U54 CUUUCUUUUCUUUUCUCACCUUACUUUACUUCCUUUCUU 78 AB030907.2b5′GCUAGUUUUC U24 G(U14)CCUCUUUUUCCGUAUUUUUUUUUUUUCCUCUUUUCUU 79 ^(a)DNAsequences were obtained from GenBank, converted to RNA sequences, andaligned to examine sequence variability. Dublicate sequences wereremoved from the alignment, and sequences are listed as [GenBankAccession #.Genotype]. ^(b)Within the 5′arm of the pU/UC tract, genotype1 nucleotide composition ranged from 12.1-44.4% purine nucleotides;genotype 2 nucleotide composition ranged from 16.7-50% purinenucleotides. ^(c)Within the 3′arm of the pU/UC tract, genotype 1nucleotide composition ranged from 0 (16 sequences)-11.6% purinenucleotides; genotype 2 nucleotide composition ranged from 5.0-11.4%purine nucleotides.

Discussion

The present data demonstrate that a nucleotide poly-uridine core of theHCV poly-U/UC tract is required for non-self recognition by RIG-I.Interspersed ribocytosine nucleotides between the poly-U sequences inthe RNA were also important for optimal RIG-I signaling to the IFN-βpromoter. The RIG-I/RNA binding studies found that RIG-I formed weakerinteractions with HCV RNAs lacking poly-U sequences, while the 34 ntpoly-U core of the poly-U/UC tract resulted in stimulating RIG-I/RNAbinding interactions. Additionally, limited-trypsin proteolysis studiesrevealed that while the RIG-I RD interacts with the 5′-ppp terminus ofHCV RNA, interactions between the helicase domain and poly-uridine HCVRNA sequences are required to activate RIG-I signaling. Finally, it wasfound that poly-U/UC RNA variants with high RIG-I signaling activityinduced significant anti-HCV responses in cultured cells and alsoinduced hepatic innate immune responses in vivo. Together, these studiesidentify long poly-uridine sequences (>U8) with interspersedribocytosines as an HCV PAMP motif that drives optimal RIG-I signaling.

Previous studies have demonstrated the importance of the 5′-ppp fornon-self recognition by RIG-I, wherein 5′-ppp was necessary but notsufficient for PAMP activity conferred by HCV RNA. The current resultsreveal the additional requirement for the U-core as a non-self signatureto demonstrate the combinatorial presentation of multiple non-selfmotifs within a PAMP RNA. These include 5′-ppp, poly-uridine sequencesand arrangements such as interspersed ribocytosine nucleotides, as wellas length and certain secondary structures, to define an RNA asnon-self. Such a requirement for combinatorial non-self recognition byRIG-I serves to provide several checkpoints for immune signaling thatprevent spurious recognition of self, thus avoiding autoimmune reactionsby requiring PAMP motifs that confer stable RIG-I interactions to induceactivation of RIG-I signaling.

RIG-I is maintained in an auto-repressed conformation in uninfectedcells. The initial step in RNA recognition and binding likely involvesRD interaction with 5′-ppp RNA due to the high affinity of the RD for5′-ppp moieties, which brings RNA structures in close contact with thehelicase domains to allow for more specific RIG-I/RNA interactions.Following ligand RNA binding, RIG-I uses ATP hydrolysis to translocatealong an RNA wherein upon engaging a PAMP motif it undergoesconformational rearrangements that release the N-terminal CARDs fordownstream ubiquitination, translocation to mitochondrial-associatedmembranes, and interaction with MAVS to drive IFN expression. RecentRIG-I structural studies have found that a V-shapedlinker/pincer/bridging domain connects the helicase domain 2 and the RD,and likely controls RIG-I conformational changes following RNA bindingthrough strong interactions with helix α17 from helicase domain 1. Inthe present study, it was found that RIG-I/RNA binding associationdifferences correlated with PAMP activity, revealing that a >8 nt poly-Ucore of the poly-U/UC tract was required to stimulate potent RIG-I/RNAbinding interactions, form contacts with the helicase domain, andactivate RIG-I signaling to the IFN-β-promoter.

These results imply a model of RIG-I interaction with the HCV PAMP RNAin which the RIG-I RD interacts with the 5′-ppp terminus of HCV ligandRNA. While kissing loop interactions between structures in the 5′ and 3′NTR of the HCV genomic strand RNA would be expected to bring the 5′-pppinto proximity to the poly-U/UC tract for non-self recognition, thesemotifs are each present in the anti-genomic strand replicationintermediate HCV RNA (5′-ppp with poly-A/AG) where they confer PAMPactivity through RIG-I recognition. This process of RIG-I binding bringsRNA sequence domains into close proximity for PAMP recognition andbinding with the helicase domain, thus providing an opportunity forRIG-I to form more stable/specific RNA contacts. ATP hydrolysis allowsRIG-I to translocate on the RNA and “scan” for HCV PAMP sequences.Following the recognition of an HCV PAMP motif, defined here aspoly-uridine sequences (>U8) with interspersed ribocytosines, RIG-Ilikely undergoes a final conformational change to activate signaling viathe CARDs and drive the innate immune response to infection.

All HCV genomes contain a poly-U sequence in the 3′ NTR; therefore,certain restrictions must exist which prevent viral evolution of thisgenomic region to mitigate RIG-I recognition. Indeed, previous studieshave reported that the poly-U/UC tract is essential for HCV RNAreplication. A minimum pU/UC core length of 26 consecutive uridinenucleotides (U26) has been indicated as required for HCV replication,while a core length of 33 uridine nucleotides has been indicated asnecessary for optimal HCV RNA amplification in cell culture. A detailedstudy revealed that in addition to the long-range kissing-loopinteractions between the NS5B SL3.2 and the 3′SL2 loops in the genomicRNA, a poly-U/UC tract with a minimum U-core length of 33 uridines (U33)is also necessary for HCV RNA replication. In addition, viral mutantswith truncated poly-U core lengths had impaired replication kinetics(U27 core) or absent replication (U7 or U16 core) until selection forlonger U-core lengths resulted in greater replication fitness. Thestrong selective pressure for a long uninterrupted poly-U nucleotidesequence within the pU/UC tract suggests that this region of the HCV RNAmay mediate essential interactions with replication factors, thusexplaining the evolution restriction of this viral genomic region. Dueto the high fitness cost, HCV is unable to prevent RIG-I recognition viagenomic sequence evolution, which makes the HCV poly-U/UC tract anoptimal target for non-self recognition. In the present study, it wasfound that deletion of the U-core resulted in the loss of PAMP activityto drive RIG-I signaling to the IFN-β-promoter. However, a poly-U coreexceeding 8 uridine nucleotides restored signaling, indicating thatRIG-I recognition of the poly-U core can occur at a U-core length belowwhat is required for efficient HCV RNA replication. This restrictionalso explains why the viral NS3/4A protease has evolved to target thedownstream signaling protein MAVS for cleavage in order to suppress theRIG-I pathway and evade restriction otherwise imposed by the innateimmune response in HCV patients.

HCV encompasses 6 major genotypes and multiple subtypes, and thisincreased viral diversity results in highly variable treatment outcomes.Standard treatment is currently limited to interferon (IFN)-basedtherapies, and although two viral protease inhibitors were recentlyapproved for use in humans, these drugs are to be applied in combinationwith IFN therapy. Overall, treatment with IFN-based therapy results inviral clearance in only 50% of infected subjects, and HCV genotypes 1aand 1b are the least responsive to standard therapies. RIG-I recognitionof HCV RNA and subsequent activation of antiviral immune responses mayinfluence the response to therapy, especially in subjects taking viralprotease inhibitors where the RIG-I pathway blockade by the viral NS3/4Aprotease would be temporarily lifted. Genetic variability within thepoly-U/UC tract of the HCV genome between different viral genotypes wasexamined using sequences available from the GenBank database. Ingeneral, HCV pU/UC genotype 1 sequences contained fewer purinenucleotides than genotype 2 sequences. In addition, nine pU/UC sequencescontained a U-core with fewer than 20 consecutive uridine nucleotides,likely representing poorly replicating genomes with decreased fitness.Further studies will help determine whether innate immune activationdiffers substantially between HCV genotypes and within patientquasispecies populations to impact the outcome of HCV infection andimmunity.

HCV evades the host immune response using multiple mechanisms, whilehost PRRs target critical regions of viral RNA or protein to suppressHCV replication. Understanding the virus-host interface regulatinginnate immunity against HCV is necessary to develop new therapies andrestrict infection. In the present study, it was found that a 5′-ppp anda poly-U core exceeding 8 uridine nucleotides within the HCV poly-U/UCtract are required for non-self recognition of HCV RNA by RIG-I. Inaddition, RIG-I recognition of the U-core within the poly-U/UC tract canactivate innate anti-HCV immune responses in vitro and hepatic innateimmune responses in vivo, thus providing a potential target forrestricting HCV infection. Similar poly-uridine molecules could be usedto induce antiviral immune responses in conjunction with IFN-based andviral protease-targeted therapies to improve HCV clearance inchronically-infected subjects. In addition, vaccine strategies thatactivate RIG-I signaling pathways may be required to induce appropriateimmune responses to RNA viruses, including HCV, which are highlysensitive to IFN and other innate antiviral ISGs in the absence of viralantagonism. Given that RIG-I can activate innate antiviral immuneresponses upon recognition of poly-uridine sequence motifs,incorporation of poly-U sequences into vaccine vectors could act as anadjuvant to mimic the natural early immune response following virusinfection.

Materials and Methods

Ethics Statement

C57BL/6 mice were housed under pathogen-free conditions in the animalfacility at the University of Washington. Experiments and procedureswere performed with approval from the University of WashingtonInstitutional Animal Care and Use Committee (IACUC; protocol number4158-01). Methods for mice use and care were performed in accordancewith the guidelines of the University of Washington Institutional AnimalCare and Use Committee, and also followed the recommendations in theGuide for the Care and Use of Laboratory Animals of the NationalInstitutes of Health.

Cells and Viruses

Huh7 cells and Huh7.5 cells were cultured in Dulbecco's modified Eaglemedium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 100μg/ml of penicillin and streptomycin. Huh7.5 cells encode a mutant RIG-Iprotein that cannot signal (Sumpter, R., Jr., et al., “RegulatingIntracellular Antiviral Defense and Permissiveness to Hepatitis C VirusRNA Replication Through a Cellular RNA Helicase, RIG-I,” J. Virol.79:2689-2699 (2005); Blight, K. J., et al., “Highly Permissive CellLines for Subgenomic and Genomic Hepatitis C Virus RNA Replication,” J.Virol. 76:13001-13014 (2002)). The hepatitis C virus (HCV) used in thesestudies was a cell culture adapted virus that was produced from thepJFH-1 HCV 2a infectious clone as previously described (Zhong, J., etal., “Robust Hepatitis C Virus Infection In Vitro,” Proc. Nat'l AcadSci. USA 102:9294-9299 (2005)).

Plasmids and Proteins

The plasmids pIFN-β-luc, pCMV-Renilla-luc (Foy, E., et al., “Regulationof Interferon Regulatory Factor-3 by the Hepatitis C Virus SerineProtease,” Science 300:1145-1148 (2003)), and pJFH-1 (Zhong, J., et al.,“Robust Hepatitis C Virus Infection In Vitro,” Proc. Nat'l Acad Sci. USA102:9294-9299 (2005); Wakita, T., et al., “Production of InfectiousHepatitis C Virus in Tissue Culture From a Cloned Viral Genome,” Nat.Med 11:791-796 (2005)) have been described. The pX-region-c4 plasmid wasgenerated by inserting an HCV Con1 X-region T7 promoter-linked PCRproduct into the pCR2.1 vector (Invitrogen) as per the manufacturer'sinstructions. Purified recombinant full-length RIG-I protein wasproduced as previously described (Jiang, F., et al., “Structural Basisof RNA Recognition and Activation by Innate Immune Receptor RIG-I,”Nature 479:423-427 (2011)).

RNA Methods.

All in vitro transcribed RNAs contain a 5′-triphosphate (5′-ppp) andthree guanine nucleotides at the 5′ end to enhance T7 polymerasetranscription. HCV X-region 5′-ppp RNA was synthesized from a T7promoter-linked PCR product generated from the pX-region-c4 plasmidusing the primers X-regionF(5′-TAATACGACTCACTATAGGTGGCTCCATCTTAGCCCTA-3′) (set forth as SEQ IDNO:80) and X-regionR (5′-ACTTGATCTGCAGAGAGGCCAGTATCA-3′) (set forth asSEQ ID NO:81). The amplified PCR product was purified by agarose gelextraction using the QIAquick gel extraction kit (Qiagen) as per themanufacturer's protocol. Full-length HCV RNA was produced from thepJFH-1 plasmid (genotype 2a) as previously described (Zhong, J., et al.,“Robust Hepatitis C Virus Infection In Vitro,” Proc. Nat'l Acad Sci. USA102:9294-9299 (2005)). All other 5′-ppp RNA products were generatedusing synthetic DNA oligonucleotide templates (Integrated DNATechnologies) and the T7 RNA polymerase as previously described(Milligan, J. F., et al., “Oligoribonucleotide Synthesis Using T7 RNAPolymerase and Synthetic DNA Templates,” Nucleic Acids Res. 15:8783-8798(1987)) using the T7 MEGAshortscript kit (Ambion) as per themanufacturer's instructions. Following in vitro transcription, DNAtemplates were removed with DNAse treatment and unincorporatednucleotides were removed from the reaction using illustra MicroSpin G-25columns (gel filtration column chromatography, GE Healthcare). RNA wasthen precipitated using ethanol and ammonium acetate as described by themanufacturer, then resuspended in nuclease-free water. RNA concentrationwas determined by absorbance using a Nanodrop spectrophotometer. RNAquality was assessed on denaturing 8 M urea polyacrylamide gels forshort RNA transcripts (50-150 nts) (not shown). Full-length HCV RNAquality was assessed on a denaturing formaldehyde-agarose gel (notshown).

Luciferase Reporter Assay

Huh7 or Huh7.5 cells were plated on 10 cm dishes, and 24 hours latercells were transfected with 5.76 μg pIFN-β-luc (firefly luciferase) and0.24 μg pCMV-Renilla-luc (Renilla luciferase) plasmids using the FuGENE6 transfection reagent and protocol (Roche). Transfected Huh7 or Huh7.5cells were incubated at 37° C. for 18 hours, then split into 48-wellplates and incubated for an additional 12 hours prior to RNAtransfection. RNA transfection was conducted in a 48-well plate formatusing the TransIT-mRNA Transfection kit (Mirus) as per themanufacturer's instructions. RNA transfection was conducted using eitherequal numbers of moles of each RNA or 350 ng RNA, depending on theexperiment. Following RNA transfection, cells were incubated anadditional 18 hours and luciferase activity was measured using theDual-Luciferase reporter assay system (Promega). All conditions andexperiments were conducted in triplicate.

EMSA

Various amounts of purified recombinant full-length RIG-I protein (0-30pmol) were mixed with 10 pmol RNA and 10 ml ATPase reaction buffer (20mM Tris-HCl, pH 8.0; 1.5 mM MgCl₂; 1.5 mM DTT). Reactions were incubatedat 37° C. for 15 minutes, then 4× native sample buffer (25 mM Tris-HCl,pH 6.8; 0.02% bromophenol blue; 60% glycerol) was added to samples.Products were separated on a 2% agarose gel (TAE, pH 7.2) and RNA wasvisualized using SYBR Gold nucleic acid stain (Invitrogen). Gel-shiftimages were analyzed using ImageJ software (National Institutes ofHealth), and RNA-protein binding curves were graphed using Prism 5software (GraphPad).

Limited Trypsin Proteolysis of RIG-I/RNA Complexes

Various amounts of RNA (0-10 pmol) were mixed with 30 pmol purifiedRIG-I protein, 2 μl of 5× reaction buffer (20 mM Tris-HCl, pH 8.0; 1.5mM MgCl₂; 1.5 mM DTT; 70 mM KCl), 0.67 μl AMP-PNP (10 mg/ml), andnuclease-free water up to 10 μl total volume. Reactions were incubatedfor 5 minutes at room temperature. Sequencing grade TPCK-treatedmodified trypsin (Promega) was added to the RIG-I/RNA mixtures at aprotease:protein ratio of 1:20 (w/w), and the reactions were incubatedat 37° C. for 15 minutes. Proteolysis was stopped by adding 0.5 ml TLCK(1 mg/ml) and incubating reactions for 5 minutes at room temperature.SDS-PAGE Laemmli sample buffer (Bio-Rad) was then added to the samplesand reaction products were analyzed by SDS-PAGE followed by silver stainusing the SilverQuest silver staining kit (Invitrogen) as per themanufacturer's instructions.

ATPase Assay

Various amounts of RNA (0-1 pmol) were mixed with 5 pmol purified RIG-Iprotein in a total of 25 μl ATPase reaction buffer (20 mM Tris-HCl, pH8.0; 1.5 mM MgCl₂; 1.5 mM DTT). Reactions were incubated at 37° C. for15 minutes, ATP (Sigma) was added to the reaction mixture at a finalconcentration of 1 mM, and the reactions were incubated at 37° C. for 15minutes. Free-phosphate concentration was determined using BIOMOL Greenreagent (Enzo Life Sciences) in a microplate format and absorbance wasmeasured at OD₆₃₀ nm.

Immunoblotting and Antibodies

Protein extracts were prepared and analyzed by immunoblotting aspreviously described (Foy, E., et al., “Regulation of InterferonRegulatory Factor-3 by the Hepatitis C Virus Serine Protease,” Science300:1145-1148 (2003)) using antibodies specific to phospho-IRF-3 Ser396(Cell Signaling Technology), IRF-3 (from A. Rustagi at University ofWashington, Seattle), RIG-I (Enzo Life Sciences), ISG56 (from G. Sen atCleveland Clinic Foundation, Cleveland), and tubulin (Sigma). Allsecondary antibodies were obtained from Jackson ImmunoResearch, andimmunoreactive bands were detected with the Amersham ECL Plus WesternBlotting Detection Reagents (GE Healthcare).

HCV Infections

Huh7 cells were plated on 48-well plates and incubated for 12-24 hoursat 37° C. Cells were transfected with 350 ng RNA using the TransIT-mRNATransfection kit (Mirus) as per the manufacturer's instructions andincubated at 37° C. for 12 hours. The transfection media was removed andthe cells were washed gently with complete DMEM. Transfected cells werethen infected with cell culture adapted JFH-1 HCV (MOI=0.1) in 100 mltotal media volume and incubated at 37° C. for 3 hours. The virusinoculum was then removed and 300 μl complete DMEM was added, and thecells were incubated at 37° C. for 48 hours. HCV-infected cellsupernatants were collected and titered on Huh7.5 cells. For the HCVtiter assay, Huh7.5 cells were plated on 48-well plates and incubatedfor 12-24 hours at 37° C., media was removed, and 100 ml of infectioussupernatants were added to cells using the following dilutions (nodilution, 1:2, 1:10, 1:100, 1:1000). Cells were incubated withsupernatants at 37° C. for 3 hours, the virus inoculum was removed andcomplete DMEM (300 μl) was added, and cells were incubated at 37° C. for48 hours. Media was then removed and the cells were washed 2 times withphosphate buffered saline (PBS). Huh7.5 cells were fixed with 4%paraformaldehyde for 30 minutes at room temperature. Cell monolayerswere permeabilized with a solution of PBS/0.2% Triton X-100 for 15minutes at room temperature, washed with PBS, and then incubated with10% fetal bovine serum in PBS for 10 minutes. After rinsing with PBS,cells were incubated with a human antiserum specific for HCV for 1 hour,washed three times with PBS, then incubated for 1 hour with donkeyanti-human-HRP secondary antibody. Cells were washed 3 times with PBS,then immunoreactive cells were visualized using the Vector VIP substratekit for peroxidase (Vector Laboratories) following the manufacturer'sinstructions. Cells were allowed to dry and focus forming units werecounted to determine HCV titers in the cell supernatants. All conditionswere conducted in triplicate.

Mice and Immunohistochemistry Staining

Mouse experiments and procedures were performed with approval from theUniversity of Washington Institutional Animal Care and Use Committee.C57BL/6 mice were transfected via intraperitoneal injection with 200 μgRNA using a lipid-based in vivo RNA transfection reagent (AltogenBiosystems), and were euthanized 8 hours later for comparativemeasurement of mRNA and protein expression. Following systemic PBSperfusion to remove contaminating blood cells, mouse livers wererecovered and fixed in 4% formalin solution for 24 hours andimmunohistochemistry staining for mouse ISG54 was performed aspreviously described (Saito, T., et al., “Innate Immunity Induced byComposition-Dependent RIG-I Recognition of Hepatitis C Virus RNA,”Nature 454:523-527 (2008)) by the Histology and Imaging Core at theUniversity of Washington.

Real-Time PCR

Mouse liver sections were collected following systemic PBS perfusion andsoaked in RNAlater reagent (Ambion). Liver sections were homogenized andRNA was extracted using the RNeasy kit (Qiagen). Synthesis of cDNA wasconducted using the iScript select cDNA synthesis kit (Bio-Rad) withboth oligo(dT) and random primers following the manufacturer'sinstructions. One-step real-time quantitative PCR was performed withSYBR Green master mix (Applied Biosystems) using an ABI PRISM 7300Real-Time PCR System. Gene specific primers for mouse IFN-β, CCL5,Ifit2, ISG15, and GAPDH were purchased from SABiosciences. Results werenormalized to the expression of mouse GAPDH mRNA.

Example 2

Summary

As described above, binding of Pathogen Associated Pattern (PAMP) RNA byRIG-I-like receptors (RLRs), RIG-I or MDA5, results in induction ofsignaling events that trigger type I interferon production and directthe innate immune response that limits pathogen infection, such aslimiting virus replication and spread. RLRs trigger interferonproduction through the activation of latent transcription factors IRF-3and NF-kB in a process that is mediated by a signalosome assembled onthe membrane-associated adaptor molecule, MAVS. Toward defining theactions of PAMP signaling by RLRs through MAVS in innate immunityagainst virus infection, it was established above in Example 1 that HCVPAMP RNA requires at least a 5′-triphosphate (5′-ppp) in associationwith a minimal poly U core sequence. The strength of RLR signaling wasincreased with the inclusion of an interspersed ribocytosine residue toprovide a poly-U/UC sequence. The poly-U/UC PAMP is immunostimulatorywhen transfected into cells or injected in mice in vivo, and thusrepresents a therapeutic agent for treatment of viral infection. Thepoly-U/UC RNA induces RIG-I signaling through the MAVS adaptor protein,but how these processes function to mechanistically limit virusinfection is not fully understood.

To further investigate the utility of a poly-U/UC sequence for use instimulating innate immune responses, additional analyses of the hostresponse induced by poly-U/UC PAMP motif from hepatitis C virus (HCV)genome RNA were conducted.

Results and Discussion

The RIG-I signaling capacities of various poly-U/UC RNA constructsderived from HCV (Con1 strain) were assayed in Huh7 cells using an IFN-βluciferase reporter assay. 10⁵ cells were co-transfected with 200 ng ofeach RNA construct along with IFN-beta luc reporter promoter plasmid.The poly-U/UC RNA constructs included: poly-U/UC, which is listed inTable 1 as “pU/UC C67U”; U24 Core, which is a derivative of thepoly-U/UC construct with a 24 residue U-core; U17 Core, which is aderivative of the poly-U/UC construct with a 17 residue U-core; U12Core, which is a derivative of the poly-U/UC construct with a 12 residueU-core; U8 Core, which is a derivative of the poly-U/UC construct withan 8 residue U-core; and Delta Core, which is a derivative of thepoly-U/UC construct but with the U-core removed. At 16 hourspost-transfection, the cells were harvested and assayed for luciferaseactivity. As illustrated in FIG. 5A, the poly-U/UC construct elicitedthe greatest induction of IFN-β luciferase reporter, with the strengthof the induction decreasing with the decrease of U-core length. IFN-βluciferase reporter was not detected with the U8 Core or Delta-Coreconstructs. This indicates that poly-U/UC RNA PAMP constructs withpoly-U core sequences over 8 nucleotides long can stimulate detectableexpression of IFN-β.

In a parallel assay, Huh7 cells similarly transfected were harvested andassayed for abundance of total IRF-3 and phospho-IRF-3 (P-IRF-3), whichis indicative of an innate immune response signaling cascade, and actinas a control. FIG. 5B illustrates that co-transfection with thepoly-U/UC RNA PAMP constructs containing poly-U core sequences over 8nucleotides long can cause the detectable phosphorylation of IRF-3.

Having established that poly-U/UC RNA PAMP constructs with poly-U coresequences over 8 nucleotides can detectably stimulate innate immuneresponse signaling, the constructs were assayed for the ability toreduce viral load in cells for a variety of different RNA viruses,namely Japanese encephalitis virus (JEV); vesicular stomatitis virus(VSV); dengue virus 2 (DV); West Nile virus (WNV); hepatitis C virus(HCV); and, respiratory syncytial virus (RSV). Cells were infected withthe indicated virus. After three hours of the infection, the cells weretreated with poly-U/UC RNA PAMP constructs of varying U-core lengths, ormock treated for control. At 72 hours post treatment, the cells wereharvested and intracellular viral RNA levels were measured usingvirus-specific RT qPCR. The results are illustrated in FIG. 6 throughFIG. 8. FIG. 6 illustrates the significant reduction in viral RNA forall viruses tested after administration of the poly-U/UC PAMP RNAconstruct. FIG. 7 illustrates the significant reduction in viral RNA forall viruses tested after administration of the poly-U/UC PAMP RNAderivative construct with a U17 core. Finally, FIG. 8 illustrates thesignificant reduction in viral RNA for all viruses tested afteradministration of the poly-U/UC PAMP RNA derivative construct with a U12core.

These data demonstrate that the tested poly-U/UC PAMP RNA constructs,with U-core sequences as short as 12 nucleotides long, have significantantiviral activity in infected cells. Moreover, consistent with the datadescribed above in Example 1, the varying lengths of the poly-U coreresult in varying strength innate response signaling. Accordingly, therelative strength of an innate immune response can be controlled throughthe rational design of poly-U/UC PAMP RNA constructs, which can beuseful to avoid over-stimulation of the innate response mechanisms.

Method and Materials

Cells and Viruses

Huh7 cells and Huh7.5 cells were cultured in Dulbecco's modified Eaglemedium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 100μg/ml of penicillin and streptomycin. Huh7.5 cells encode a mutant RIG-Iprotein that cannot signal. The hepatitis C virus (HCV) used in thesestudies was a cell culture adapted virus that was produced from thepJFH-1 HCV 2a infectious clone.

Plasmids and Proteins

The plasmids pIFN-β-luc, pCMV-Renilla-luc, and pJFH-1 encode humanIFN-beta promoter driving firefly luciferase expression, and a CMVpromoter driving Renilla luciferase expression, respectively. ThepX-region c4 plasmid was generated by inserting an HCV Con1 X-region T7promoter-linked PCR product into the pCR2.1 vector (Invitrogen) as perthe manufacturer's instructions.

RNA Methods

All in vitro transcribed RNAs contain a 5′-triphosphate (5′-ppp) andthree guanine nucleotides at the 5′ end to enhance T7 polymerasetranscription. For negative control, HCV X-region 5′-ppp RNA wassynthesized from a T7 promoter-linked PCR product generated from thepX-region c4 plasmid using the primers X-regionF and X-regionR,described above in Example 1 and set forth herein as SEQ ID NOS:80 and81, respectively. The amplified PCR product was purified by agarose gelextraction using the QIAquick kit (Qiagen) as per the manufacturer'sprotocol. 5′-ppp RNA products were generated using synthetic DNAoligonucleotide templates (Integrated DNA Technologies) and the T7 RNApolymerase as described by the manufacturer using the T7 MEGAshortscriptkit (Ambion). The various derivatives of the poly-U/UC RNA constructsincluded, namely the U12, U17, and U24 core RNA constructs differed fromthe poly-U/UC RNA (“pU/UC C67U” in Table 1) by virtue of shorter poly-Ucore sequences, as indicated, and with a C21 nucleotide at the 3′ end.Following in vitro transcription, DNA templates were removed with DNAsetreatment and unincorporated nucleotides were removed from the reactionusing illustra MicroSpin G-25 columns (gel filtration columnchromatography, GE Healthcare). RNA was then precipitated using ethanoland ammonium acetate as described by the manufacturer, then resuspendedin nuclease-free water. RNA concentration was determined by absorbanceusing a Nanodrop spectrophotometer. RNA quality was assessed ondenaturing 8M urea polyacrylamide gels for short RNA transcripts (50-150bp). Full-length HCV RNA quality was assessed on a denaturingformaldehyde-agarose gel.

Luciferase Reporter Assay

Huh7 cells were plated on 10 cm dishes, and 24 hours later cells weretransfected with 5.76 μg pIFN-β-luc (firefly luciferase) and 0.24 μgpCMV-Renilla-luc (Renilla luciferase) plasmids using the FuGENE 6transfection reagent and protocol (Roche). Transfected Huh7 or Huh7.5cells were incubated at 37° C. for 18 hours, then split into 48-wellplates and incubated for an additional 12 hours prior to RNAtransfection. RNA transfection was conducted in a 48-well plate formatusing the TransIT-mRNA Transfection kit (Mirus) as per themanufacturer's instructions. RNA transfection was conducted using eitherequal numbers of moles of each RNA or 350 ng RNA, depending on theexperiment. Following RNA transfection, cells were incubated anadditional 18 hours and luciferase activity was measured using theDual-Luciferase reporter assay system (Promega). All conditions andexperiments were conducted in triplicate.

Immunoblotting and Antibodies

Protein extracts were prepared and analyzed by immunoblotting aspreviously described in Schnell, G., et al., “Uridine Composition of thePolu-U/UC Tract of HCV RNA Defines Non-Self Recognition by RIG-I,” PLOSPathogens 8(8):e1002839 (2012) using antibodies specific tophospho-IRF-3 Ser396 (Cell Signaling Technology), IRF-3 (from A. Rustagiat University of Washington, Seattle), and actin (Sigma). All secondaryantibodies were obtained from Jackson ImmunoResearch, and immunoreactivebands were detected with the Amersham ECL Plus Western BlottingDetection Reagents (GE Healthcare).

Virus Infection

Respiratory syncytial virus (RSV) was prepared from HeLa cells usingvirus stocks obtained from M. E. Peeples (Ohio State University).Infected HeLa cells and culture medium were collected at 48 hrspost-infection. The cell pellet was resuspended in PBS supplemented with2 mM EDTA, and lysed by successive cycles of freezing and thawing.Cellular debris was removed by centrifugation, and the supernatantpooled with the previously collected culture medium. Virus wasconcentrated with the addition of 50% (v/v) polyethylene glycol (PEG,FW=8000) in NTE buffer (150 mM NaCl, 50 mM Tris base pH 7.2, 10 mM EDTA)to a final concentration of 10% (v/v) PEG, followed by centrifugation at10,000 rpm. The virus pellet was reconstituted in 20% sucrose in NTEbuffer, and further purified by sedimentation using a discontinuousgradient of 35 and 60% (w/v) sucrose in NTE buffer that was centrifugedat 37000 rpm for 1 hr at 4° C. The purified virus stock was titrated onHeLa cells and assayed for focus forming unitus (FFU) at 48 hrspost-infection. Vesicular stomatitis virus (VSV) was a gift from Dr.Phillip Marcus (University of Connecticut). Dengue virus type 2 was agift from Lee Gehrke (Massachusetts Institute of Technology and HarvardMedical School). Hepatitis C virus (HCV) was produced from the JFH1 HCVinfectious clone as described by Saito, T., et al., “Innate ImmunityInduced by Composition-Dependent RIG-I Recognition of Hepatitis C VirusRNA,” Nature 454:523-527 (2008). Japanese Encephalitis virus (JEV;strain 14-14-2) was a gift from Michael Diamond, (WashingtonUniversity). West Nile virus strain TX-02 was isolated and prepared asdescribed in Keller, B. C., et al., “Resistance to alpha/beta interferonis a determinant of West Nile virus replication fitness and virulence,”J. Virol. 80:9424-9434 (2006). Virus infections were conducted asfollows: Cells were infected with virus at multiplicity of infection(MOI) of 1.0. 5×10⁵ cells were cultured in a 6 cm dish in complete mediawith 10% fetal bovine serum (DME). Cells were transfected withtransfection reagent alone (media control) or with the indicated PAMPRNA (1 ug/dish). 16 hours later the media was removed and the cells wererinsed 3 times with fresh serum-free media. After the final rinse, theindicated virus in serum-free media was added to the culture, afterwhich the cultures were incubated for 4 hr at 37° C. After 4 hr themedia was removed and replaced with DME, and the cells were cultured foran additional 48 hrs. Cells were then harvested by removing the media,washing the cell monolayer in PBS and scrapping the cells off of theculture dish. Cell pellets were collected and subjected to freeze-thawfor cell lysis, and cell extracts were prepared for immunoblot analysisor RT-qPCR analysis.

PCR

RNA was isolated from cell extracts using the RNA isolation kit fromQiagen. Synthesis of cDNA was conducted using the iScript select cDNAsynthesis kit (Bio-Rad) with both oligo(dT) and random primers followingthe manufacturer's instructions. One-step real-time quantitative PCR wasperformed with SYBR Green master mix (Applied Biosystems) using an ABIPRISM 7300 Real-Time PCR System. Gene specific primers for human ormouse GAPDH were purchased from SABiosciences. Virus-specific primersfor HCV, reovirus, JEV, DV, VSV, and RSV were from Loo et al., (Loo,Y.-M., et al., “Distinct RIG-I and MDA5 signaling by RNA viruses ininnate immunity,” J. Virol. 82:335-345 (2008)). Results were normalizedto the expression of mouse GAPDH mRNA.

Statistics

Data sets were analyzed by Student's T test for assessment ofsignificant differences. P value <0.5 represents statisticallysignificant differences.

Example 3

Summary

As described above, RLRs serve to recognize and bind to viral productRNA to identify nonself for immune response induction. RIG-I is bestunderstood in this capacity. In the case of hepatitis C virus (HCV)infection, RIG-I recognizes and binds to HCV genome RNA throughrecognition of free 5′ triphosphate (5′-ppp) and thepoly-uridine/cytosine (poly-U/UC) present in the viral RNA 3′nontranslated region (NTR) (collectively known as the poly-U/UC PAMP).The poly-U/UC PAMP is immunostimulatory when transfected into cells orinjected in mice in vivo, and thus represents a therapeutic agent fortreatment of viral infection. It is demonstrated in Examples 1 and 2that poly-U/UC PAMPs with a poly-U core over 8 nucleotides in length canhave an innate immunostimulatory effect and can confer anti-viral effectin vitro and in vivo.

This Example describes additional assays to determine the utility andefficacy for various poly-U/UC RNA constructs to induce innate responsesin vivo. It was determined from in vivo assays that the poly-U/UC RNAconstructs induce innate immune responses through MAVS/RLR-mediatedsignaling and protect mice from lethal West Nile virus challenge.

Results and Discussion

As described in Examples 1 and 2, a polyU/UC region from the hepatitis Cvirus (HCV) RNA genome was discovered as a PAMP that specificallyengages and triggers RIG-I-dependent signaling of innate antiviralimmunity. The present study demonstrates the selective triggering ofRIG-I/MAVS signaling using the HCV-derived poly-U/UC PAMP RNA constructsprotects mice from virus challenge. Mice injected with the HCV PAMPexpress various interferon stimulated genes (ISG) in tissues includingthe liver as compared to mice injected with either PBS alone or theantagonistic X region RNA from the HCV genome (FIGS. 1A and 1B).Specifically, FIG. 9A illustrates an immunohistological stain of livertissue from wt MAVS mice or mice with a double negative mutant for MAVS.The figure demonstrates the detection of ISG54 expression only in the wtMAVS mice that were administered 200 μg poly-U/UC RNA (as describedabove in Example 2). FIG. 9B is an immunoblot that demonstrates theexpression of ISG54, ISG56, and ISG15 only in liver cells from wt MAVSmice that had been administered the poly-U/UC RNA. Mice injected withthe HCV poly-U/UC RNA PAMP further exhibit elevated levels of type Iinterferon in their serum (FIG. 9C and data not shown). This signalingis strictly dependent on MAVS as mice lacking MAVS express little or noISG in the liver and only background levels of type I interferon isdetectable in the serum.

In view of the above, it was hypothesized that the HCV PAMP RNA andother RLR agonists that can specifically trigger RLR signaling might beuseful as adjuvants or antiviral therapies. To test this hypothesis, theeffect on administration of HCV PAMP RNA constructs on in vivo viralburden was assayed. SCID/beige-Alb/uPA chimeric mice were transplantedwith human primary hepatocytes. After confirming expression of humanalbumin, mice received 100 mL HCV-positive patient serum, and wereadministered thereafter at days 11, 13, and 15 post-infection with PBSalone or 150 mg polyU/UC (PAMP) RNA or control xRNA by hydrodynamic IVinjections via the tail vein. Mice were bled at 7 days post-infectionand at 14 days post-infection to establish viremia levels for qPCRstudies to establish pre-treatment viremia and viremia during the courseof treatment. FIG. 10 illustrates the log scale difference in HCVviremia levels between days 14 and 7, indicating that administration ofthe polyU/UC PAMP RNA construct resulted in a significant reduction inviral burden in vivo. A similar assay was performed, but wherein themice were bled at day 10 post-infection (to establish a pre-treatmentviremia baseline) and at days 14 and 15 post-infection (to establishviremia levels during and post treatment). FIGS. 11A and 11B illustratesimilar patterns as in FIG. 10, wherein administration of the polyU/UCPAMP RNA construct resulted in significant reduction of viral burden invivo.

Further investigations were conducted to determine whetheradministration of the polyU/UC PAMP RNA construct could conferprotection in vivo to other viruses. In a first assay, C57Bl6 mice wereinfected with West Nile virus (WNV), and thereafter at days 1.5, 3, and5, mice were given 100 mg polyU/UC RNA or control XRNA by IP, andmonitored daily for body weight and clinical scores. FIG. 12 is asurvivorship plot of the mice that demonstrates that the poly-U/UC PAMPRNA construct, but not the XRNA construct, protected the mice againstdisease and/or disease severity of WNV infection. In a second assay,wildtype and double negative MAVS mutant mice were challenged with WestNile virus. The mice were provided the HCV polyU/UC PAMP RNA PAMPconstruct, the antagonistic HCV RNA of equivalent length (XRNA) or PBSalone. The wildtype mice that were provided the HCV PAMP RNA demonstratereduced virus burden in the spleen and fewer succumb to West Nile virusinfection as compared to mice injected with PBS alone or mice injectedwith XRNA (FIGS. 13A and 13D). Mice that survive the virus challengealso exhibit less severe weight loss and clinical scores when providedthe HCV PAMP RNA, suggesting that HCV PAMP RNA has a protective functionagainst WNV challenge (FIGS. 13E and 13F). In contrast, MAVS deficientmice remain highly susceptible to West Nile virus challenge regardlessof whether they are provided the HCV PAMP RNA, suggesting that the HCVPAMP RNA provided no protection to the mice. The data thus demonstratesthat the HCV PAMP RNA is capable of triggering innate immune response,thereby conferring protection against infections of various viruses. Thedata also emphasizes the important nature of MAVS in signaling innateantiviral immunity and protection from virus infection.

Numerous studies have shown that MAVS is essential for the control ofvirus infection and immunity in vivo. In this study, it is demonstratedthat the HCV polyU/UC RNA PAMP construct can selectively triggerMAVS-dependent signaling as a RIG-I-specific agonist. Not only did theadministration of the polyU/UC PAMP RNA construct increase the averagesurvival rate of mice, it also reduced weight loss and severity ofclinical symptoms that is associated with WNV infections as compared toadministration of PBS or an antagonist RNA. This data confirms previoussuggestions that agents that the polyU/UC PAMP RNA can driveRIG-I/MAVS-dependent signaling and suggest that the constructs are goodtherapeutic platform for the development of novel antiviral treatmentsor adjuvants to enhance vaccine immunity.

Methods and Materials

Cell Culture and Viruses

Huh7, HEK293 and the replicon cells Huh7-HCV K2040 and Huh7-HCV A7,which harbor self-replicating subgenomic HCV genotype 1b RNA, werecultured according to standard techniques. To create non-targetingvector control (NTV) and MAVS knockdown cells, Huh7 were transduced witha lentivirus expressing non-specific control shRNA (previously verifiednot to target any known human or mouse sequences) or a lentivirusexpressing shRNA that specifically targets MAVS according tomanufacturer's recommendations. Stable cells were selected andpropagated in complete media supplemented with hygromycin. Lentivirusstocks were acquired from the Sigma-Aldrich Mission shRNA collection.The shRNA sequences were encoded by the lentivirus (MAVS knockdown).Stocks of Cantell strain Sendai virus were acquired from Charles RiverLaboratory. Cell culture adapted JFH1 genotype 2A HCV and WNV-Tx werepropagated and infectivity measured as described elsewhere(Fredericksen, B. L., et al. “The host response to West Nile Virusinfection limits viral spread through the activation of the interferonregulatory factor 3 pathway,” J. Virol. 78(14): 7737-7747 (2004); Loo,Y. M., et al. “Viral and therapeutic control of IFN-beta promoterstimulator 1 during hepatitis C virus infection,” Proc. Nat'l Acad. Sci.U.S.A. 103(15): 6001-6006 (2006)).

Plasmids and Transfections

The following plasmids used in this study have been described elsewhere:pEFTak MAVS and C508Y mutant, pcDNA3.1 Myc MAVS, HA-NEMO, reporterplasmids (pIFNb-Luc, PRDii-Luc, F3-Luc, Renilla-Luc), IRF3-5D, andpEFTak N-RIG (Foy, E., et al. “Regulation of interferon regulatoryfactor-3 by the hepatitis C virus serine protease,” Science300(5622):1145-1148 (2003); Sumpter, R., Jr., et al. “Regulatingintracellular antiviral defense and permissiveness to hepatitis C virusRNA replication through a cellular RNA helicase, RIG-I,” J. Virol.79(5):2689-2699 (2005); Loo, Y. M., et al. 2006). DNA transfections wereperformed using FuGene 6 (Roche) or Lipofectamine 2000 (Invitrogen) asrecommended by the manufacturer. Promoter luciferase reporter assayswere conducted as described (Foy, Li et al. 2003). HCV XRNA and polyU/UCPAMP RNA (“pU/UC C67U” in Table 1) were generated using the MegaScriptin vitro transcription kit (Ambion) as described below, above inExamples 1 and 2, and elsewhere (Saito, T., et al., “Innate immunityinduced by composition-dependent RIG-I recognition of hepatitis C virusRNA,” Nature 454(7203): 523-527 (2008)).

RNA Methods

All in vitro transcribed RNAs contain a 5′-triphosphate (5′-ppp) andthree guanine nucleotides at the 5′ end to enhance T7 polymerasetranscription. HCV X-region 5′-ppp RNA was synthesized from a T7promoter-linked PCR product generated from the pX-region c4 plasmidusing the primers X-regionF and X-regionR, described above in Example 1and set forth herein as SEQ ID NOS:80 and 81, respectively. Theamplified PCR product was purified by agarose gel extraction using theQIAquick kit (Qiagen) as per the manufacturer's protocol. Full-lengthHCV RNA was produced from the pJFH-1 plasmid (genotype 2a) as previouslydescribed (Saito et al., 2008). All other 5′-ppp RNA products weregenerated using synthetic DNA oligonucleotide templates (Integrated DNATechnologies) and the T7 RNA polymerase (as described by Saito et al.,2008) using the T7 MEGAshortscript kit (Ambion) as per themanufacturer's instructions. Following in vitro transcription, DNAtemplates were removed with DNAse treatment and unincorporatednucleotides were removed from the reaction using Illustra MicroSpin G-25columns (gel filtration column chromatography, GE Healthcare). RNA wasthen precipitated using ethanol and ammonium acetate as described by themanufacturer, then resuspended in nuclease-free water. RNA concentrationwas determined by absorbance using a Nanodrop spectrophotometer. RNAquality was assessed on denaturing 8M urea polyacrylamide gels for shortRNA transcripts (50-150 bp). Full-length HCV RNA quality was assessed ona denaturing formaldehyde-agarose gel.

Luciferase Reporter Assay

Huh7 or Huh7.5 cells were plated on 10 cm dishes, and 24 hours latercells were transfected with 5.76 μg pIFN-β-luc (firefly luciferase) and0.24 μg pCMV-Renilla-luc (Renilla luciferase) plasmids using the FuGENE6 transfection reagent and protocol (Roche). Transfected Huh7 or Huh7.5cells were incubated at 37° C. for 18 hours, then split into 48-wellplates and incubated for an additional 12 hours prior to RNAtransfection. RNA transfection was conducted in a 48-well plate formatusing the TransIT-mRNA Transfection kit (Mirus) as per themanufacturer's instructions. RNA transfection was conducted using eitherequal numbers of moles of each RNA or 350 ng RNA, depending on theexperiment. Following RNA transfection, cells were incubated anadditional 18 hours and luciferase activity was measured using theDual-Luciferase reporter assay system (Promega). All conditions andexperiments were conducted in triplicate.

Antibodies

The following antibodies were used in this study: anti-Flag M2(Sigma-Aldrich), anti-Myc 9E10 (AbCam), anti-Myc (AbCam), anti-Cardif(for MAVS; Axxora), anti-ISG54 and ISG56 (G. Sen), anti-IRF-3 (M.David), anti-IRF-3 phospho (Cell Signaling), anti-ISG15 (A. Haas),anti-HA (Sigma), anti-IKKe (Santa Cruz), anti-TANK (Santa Cruz),anti-TBK1 (Santa Cruz), anti-TRAF6 (Sigma), anti-NEMO (Sigma),anti-COX-IV (Molecular Probes), anti-actin (Sigma), anti-tubulin(Sigma), and polyclonal human anti-HCV (hyperimmune sera from anHCV-infected patient). Mitotracker and DAPI were acquired from Molecularprobes.

MAVS Knockout Mice

MAVS knockout mice in the C57Bl/6 background were created usingconventional methods at inGenious Targeting Laboratory, Inc. usingC57Bl/6 ES cells by replacing exons 2-3 of MAVS (containing the ATGstart codon) with a neomycin cassette. Deletion of exons 2-3 wasverified by Southern blot (not shown). Northern blot and qPCR analysesconfirm that cells from knockout mice do not express MAVS mRNA. Mice aregenotyped using primers 5′-ATGGGATCGGCCATTGAACAAGATC-3′ (set forth asSEQ ID NO:82), 5′-CACCCAGCCACCAGAGTCCCCAG-3′ (set forth as SEQ IDNO:83), and 5′-CCCTGCCTCCTGTCTAAGGAAGG-3′(set forth as SEQ ID NO:84) fordetection of both the wildtype and mutant alleles.

Poly-U/UC PAMP RNA Transfection and Virus Challenge In Vivo

C57Bl/6 mice of matching age and gender were used as wildtype controlsin all experiments. PBS, HCV XRNA or HCV polyU/UC PAMP RNA mixed inlipid-based In Vivo transfection reagent (Altogen Biosystems) wasinjected by the intraperitoneal route as recommended by themanufacturer. Mice were euthanized at indicated time points to collectblood and tissues. For liver immunohistochemistry, perfused livers werefixed in 4% paraformaldehyde and imbedded in paraffin. 5 nm sectionswere affixed to charged slides, and processed for immunohistochemistryat the UW Core facility. Serum cytokine levels were measured using theVerikine Interferon-β ELISA kit (PBL). For infection experiments, micewere injected in one footpad with 1×10² PFU WNV-TX. Mice were provided200 μg of either HCV XRNA or polyU/UC PAMP RNA by IP at days 1, 3, 5,and 7 post-infection. Mice were weighed daily and monitored for changesin clinical scores as described previously (Suthar, M. S., et al., “MAVSis essential for the control of West Nile virus infection and immunity,”PLoS Pathogens 6(2):e1000757 (2010)). All mouse breeding and experimentswere conducted in specific pathogens-free facilities in strictaccordance to protocols approved by the University of Washington IACUC.

Immunoprecipitations

HEK293 cells were co-transfected with a Flag-tagged bait construct andeither a Myc- or HA-tagged target construct using FuGene 6 (Roche). Cellpellets were collected 24 hrs after transfection and theimmunoprecipitations performed using Flag antibody-coated agarose beads(Sigma-Aldrich) in RIPA buffer (50 mM Tris-HCl pH 7.4, 100 mM NaCl, 1%Triton-x100, 0.5% sodium deoxycholate). Precipitates were analyzed bySDS-PAGE and immunoblotting using standard techniques.

RNA Analysis

Total RNA was extracted from cultured cells using the RNeasy kit(Qiagen). HCV copy number was measured from triplicate reactions by realtime-quantitative PCR (qPCR) as previously described using an AppliedBiosystems 7300.

Example 4

Summary

As described above, the HCV-derived poly-U/UC PAMP RNA can driveRIG-I/MAVS-dependent signaling and mediate an innate immune response.Furthermore, it was demonstrated above that administration of theHCV-derived poly-U/UC PAMP RNA construct conferred protection againstdifferent viral infections in mice. The present Example addresses theutility of using a HCV-derived poly-U/UC PAMP RNA, as described above,as an adjuvant to enhance the efficacy vaccine components in protectingagainst viral disease.

Introduction

West Nile virus (WNV) is a neurotropic flavivirus that constitutes theleading cause of mosquito-borne and epidemic encephalitis in humans inthe United States. WNV is a member of the family Flaviviridae andcarries a single-stranded positive-sense RNA genome of approximately 11kb in length consisting of a single open reading frame that istranslated as a polyprotein to generate ten viral proteins. Lineage 1WNV strains represent emerging viruses that associate with outbreaks ofencephalitis and meningitis in Europe, the Middle East, and now in NorthAmerica, whereas lineage 2 strains are typically nonpathogenic,nonemergent and geographically confined to the African subcontinent andMadagascar. More recently, lineage 1 WNV associated infections haveshifted from causing disease in young children, elderly, and theimmunocompromised to afflicting healthy young adults, indicating thatvirulence occurs independently of immune senescence or immunedeficiencies associated with aging. Pathogenic lineage 2 WNV variantshave recently emerged in Europe, causing significant WNV-induced diseasein humans. The increase in virulence of lineage 1 and 2 strains, coupledwith a lack of a vaccine or therapeutic agents continues to present WNVas a significant public health threat.

The host innate immune response is the first line of defense duringvirus infection and is responsible for deterring virus replication andspread within the host. Lineage 1 WNV-TX, but not lineage 2 WNV-MAD, hasbeen shown to inhibit type I IFN-induced phosphorylation of STAT1 andSTAT2 by blocking the activity of the IFN receptor-associated kinase,Tyk2. It was found that WNV-TX also blocks an IKKε-dependentphosphorylation event on STAT1, resulting in a temporal regulation ofSTAT1 phosphorylation and subsequent expression of interferon-stimulatedgenes that are essential for controlling WNV infection. However, studiesto identify specific viral determinants that regulate WNV inhibition ofIFN-mediated signaling have been hindered by the lack of appropriatereagents.

Results and Discussion

As described previously in Suthar, M. S., et al., “Infectious Clones ofNovel Lineage 1 and Lineage 2 West Nile Virus Strains WNV-TX02 andMNV-Madagascar,” J. Virol. 86(14):7704-7709 (2012), incorporated hereinby reference in its entirety, to facilitate viral genetic studies ofWNV/host interactions that control infection and immunity, a novelinfectious clone (i.c.) of WNV-TX (strain TX 2002-HC) and a clone ofWNV-MAD (strain Madagascar-AgMg798) were generated. The design of eachinfectious clone is based on a two-plasmid reverse genetics system. Inthis system, the structural and nonstructural genes are broken into twoplasmids (pAB and pCG, respectively). This system allows for overcominglimitations imposed by genetic instability during plasmid propagation inbacterial hosts, problems due to difficult low-yield plasmidpreparations, as well as limitations due to a lack of restriction sitesfor cDNA insertion in single-plasmid cloning schemes. To generate theinfectious clones, the coding sequences of NY99 strain 382-99 werereplaced with either WNV-TX or WNV-MAD in the respective plasmids. Thesequence of the viral RNA 5′ UTR is conserved among WNV-NY99, WNV-TX,and WNV-MAD. The 3′ UTR sequence of the viral RNA is conserved betweenWNV-NY99 and WNV-TX, but not WNV-MAD (73.4% sequence similarity). Forgenerating the WNV-TX infectious clone, three amino acid coding changeswithin the sequence of WNV-NY99 (strain 382-99) were replaced withWNV-TX. The WNV-MAD infectious clone was synthesized (Genscript,Piscataway, N.J.) with nucleotides (nt) 1-2500 (encoding the structuralgenes) inserted in a pUC19 vector (Genscript) and nt 2494-10396 (withthe 3′UTR (nt 10397-11031) from WNV-TX) in a pCCI vector (Genscript). Toallow for full-length clone assembly, a unique NgoMIV restriction sitewas engineered (nt A2496C, A2498G) that did not alter the NS1 amino acidcoding sequence. Infectious WNV-TX and WNV-MAD RNA was successfullyprepared from their respective two-plasmid infectious clones, introducedin BHK-21 cells by electroporation, and viral supernatants recovered andaliquoted as the primary working stocks for phenotypic analysis. The twonovel strains were characterized to confirm that they retainedcharacteristics of their parental strains. The resulting WNV-TX andWNV-MAD infectious clones, which differ in their abilities to inhibittype I IFN signaling, provide a platform for identification of novelviral determinants in regulating type I IFN signaling and responses.

The novel avirulent WNV-MAD infectious clone can serve as alive-attenuated vaccine strain and permit the present study to addressthe capacity of the poly-U/UC PAMP RNA constructs to enhance hostresponses against infections pathogens when administered in associationwith vaccine components.

FIG. 14A illustrates the vaccination schedule performed in the presentstudy. At 21 days prior to C57Bl6 mice (5-6 weeks old) were vaccinatedwith 100 mg polyU/UC PAMP RNA alone, 0.25 mg UV-inactivated WNV (MADclone) +PBS, or 0.25 mg UV-inactivated WNV mixed with 100 mg polyU/UCPAMP RNA or xRNA control. At “Day 0” the mice were challenged with 1000pfu WNV (TX-02 clone) administered via subcutaneous injection of theleft footpad. Mice were monitored daily for body weight and clinicalscores. FIG. 14B illustrates the survival patterns for the groups ofmice after challenge with the pathogenic WNZ (Tx) strain. Differencesshown between WNZ (MAD clone) vaccine +poly-U/UC PAMP RNA and othertreatment groups were significant (P<0.03), with all individualssurviving to the end of the observation period. This demonstrates thatpoly-U/UC RNA is an effective vaccine adjuvant for enhancement ofprotection by virus vaccines.

Methods and Materials

Preparation attenuated and pathogenic virus strains for the challengestudies are described in Suthar, M. S., et al., “Infectious Clones ofNovel Lineage 1 and Lineage 2 West Nile Virus Strains WNV-TX02 andMNV-Madagascar,” J. Virol. 86(14):7704-7709 (2012), incorporated hereinby reference in its entirety.

UV-inactivated stocks of WNV-MAD were prepared by irradiating the virususing a Spectrolinker XL-1000 at 120 mJ/cm² for 30 minutes. Infectivityof virus stocks was verified by plaque assay on VERO cells, and proteinconcentration of the stocks quantified by BCA assay (Thermo ScientificPierce) using a BSA standard according to manufacturer's instructions.

In Vivo Vaccine-Challenge Studies

5-6 week old C57Bl/6 mice were vaccinated subcutaneously with 100 ugpolyU/UC RNA alone, 0.25 ug UV-inactivated WNV-MAD+PBS or 0.25 ugUV-inactivated WNV-MAD mixed with 100 ug of polyU/UC RNA or xRNAcontrol. 21 days after vaccination, mice were challenged with 1000 pfuWNV (Tx-02) administered via subcutaneous injection of the left footpad.Mice were monitored daily for body weight and clinical scores. Mice thatexhibit 20% or greater weight loss or clinical symptoms greater than 5(moribund) were euthanized according to UW IACUC approved protocols.Clinical symptoms were determined according to the following scoringsystem:

0: Healthy mouse

1: Ruffled fur, lethargy, hunched posture, no paresis

2: Very mild to mild paresis affecting one or both hind limbs

3: Frank paresis involving one hind limb or mild paresis in both hindlimbs, conjunctivitis

4: Severe paresis involving both hind limbs but mouse retains feelingand is possibly limbic

5: True paresis

6: Moribund

7: Euthanized mouse

Example 5

Summary

This Example describes characterization of Hepatitis C 3′ poly-U/UCregions from isolates obtained from human subjects. The data confirmsthe variability of the length of poly-U core among wild isolates andindicates that short poly-U core lengths are likely to contribute toRIG-I signaling.

RESULTS AND DISCUSSION

Hepatitis C RNA was sequenced from biological samples obtained fromhuman patients. The 3′ poly-U/UC region for the isolates are presentedbelow in Table 3. Several isolates contained poly-U core sequences under20 nucleotides in length, indicating that such PAMPs exist in nature.Additional studies can be performed on such PAMPs to determine theircapacities to bind RIG-1 and stimulate subsequent innate immuneresponses through RIG-I/MAVS-dependent signaling. PAMP sequences withvarious length and short U-core regions have the ability to “tune” theinnate immune response to specific applications. For example, a robustresponse might be required to induce protective immunity against aspecific pathogen while a reduced response might be required to triggerlower level innate immunity against specific microbial agents wheremicrobial-induced inflammation has been triggered during infection.

TABLE 3 Sequence of 3′poly-U/UC regions for HCV isolates from human subjects. plasmid Geno- U-SEQ # Founder type 5′ arm Core 3′ arm ID NO: PAMP HCV Con1 1bGGCCAUCCUG(U7)CCC U34 CUCC(U9)CCUC(U7)CCUUUUCUUUCCUUU 85 (U11)C 17169055TF.UC1 3a CCAUUUUUC U13 GUUUG(U16)CUUUCCUUCUUUCCUGACUUUU 86AAUUUUCCUUCUUA 1717 9055TF.UC2 3a CCAUUUUUC U49GUUUG(U17)CUUUCCUUCUUUCCUGACUUUU 87 AAUUUUCCUUCUUA 1713 10021TF.UC1 1aGGCCAUUUCCUG U33 ACCCUUUUUUCUC(U12)CCUUCUUCUUUAAU 88 1714 10021TF.UC2 1aGGCCAUUUCCUG U18 ACCCUUUUUUCUC(U17)CCUUCUUCUUUAAU 89 1720 10025TF.UC1 1aGGCCAUUUUCUG U14 AUUUUCUUUAAU 90 1721 10025TF.UC2 1a GGCCAUUUUC(U10)CUCU18 AUUUUCUUUAAU 91 1722 10025TF.UC3 1a GGCCAUUUUCUG U20CC(U12)CCUC(U20)AUUUUCUUUAAU 92 1723 10025TF.UC4 1a GGCCAUUUUCUG(U12)CU17 CCUUUUUUUUUCUC(U14)AUUUUCUUUAAU 93 1715 10051TF.UC1 1b GGCCAUCCUGU24 C(U17)CUUUUUCC(U13)AUUUUCUUCUUU 94 1725 105431TF.UC1 4a GGUCCUAAGU13 CUUCCUUCCUUCUUUCCUUUUCUAAUUUUCCUUCUUU 95 1726 105431TF2.UC1 4aGGUCCUAAGUUG U15 CCUUCCUUCUUUCCCUUUUCUAAUUUUCCUUCUUU 96 1727105431TF2.UC2 4a GGUCCUAAGUUG U23CCUUUCCUUCCUUCUUUCCUUUUCUAAUUUUCCUUCUUU 97 1718 110069TF1.UC1 1aGGCCAUUUCUG U41 GUUUVVUUVUUUUUVVUUUUV(U11)CUCCCUUUAAU 98 1719110069TF1.UC2 1a GGCCAUUUCUG U14 GUUUCCUUCUUUUUCCUUUUC(U13)CUCCCUUUAAU99

While illustrative embodiments have been illustrated and described, itwill be appreciated that various changes can be made therein withoutdeparting from the spirit and scope of the invention.

The invention claimed is:
 1. An isolated nucleic acid molecule with anon-naturally occurring sequence, the isolated nucleic acid comprising:a 5′-arm region comprising a terminal triphosphate; a poly-uracil corecomprising at least 8 contiguous uracil residues; and a 3′-arm regioncomprising at least 8 nucleic acid residues, wherein the 5′-most nucleicacid residue of the 3′-arm region is not a uracil and wherein the 3′-armregion is at least 80% uracil residues, wherein the 5′-arm region andthe poly-uracil core do not naturally occur contiguously together in aHepatitis C virus.
 2. The isolated nucleic acid molecule of claim 1,wherein the poly-uracil core consists of between 8 and 30 uracilresidues.
 3. The isolated nucleic acid molecule of claim 1, wherein the5′-most nucleic acid residue of the 3′-arm region is a cytosine residueor a guanine residue.
 4. The isolated nucleic acid molecule of claim 1,wherein the 3′-arm region is at least 90% uracil residues.
 5. Theisolated nucleic acid molecule of claim 1, wherein the 3′-arm regioncomprises at least 7 contiguous uracil residues.
 6. The isolated nucleicacid molecule of claim 1, wherein the 5′-arm region further comprisesone or more nucleic acid residues disposed between the terminaltriphosphate and the poly-uracil core.
 7. The isolated nucleic acidmolecule of claim 1, wherein the nucleic acid molecule is capable ofinducing retinoic acid-inducible gene I (RIG-I)-like receptor (RLR)activation.
 8. The isolated nucleic acid molecule of claim 7, whereinthe RLR is RIG-I.
 9. A pharmaceutical composition comprising theisolated nucleic acid molecule of claim 1 and an acceptable carrier. 10.The pharmaceutical composition of claim 9, further comprising a viralantigen, a bacterial antigen, a protozoal antigen, a fungal antigen,and/or a helminth antigen, or an attenuated, inactivated, or killedvirus, bacterium, protozoan, fungus, and/or helminth.
 11. Thepharmaceutical composition of claim 9, further comprising an anti-viraltherapeutic, an anti-bacterial therapeutic, an anti-protozoaltherapeutic, an anti-fungal therapeutic, an anti-helminth therapeutic,and/or an adjuvant.
 12. The isolated nucleic acid molecule of claim 1,wherein the 5′-arm region consists of the terminal triphosphate, andwherein the terminal triphosphate is linked directly to the 5′-end ofthe poly-uracil core.